In this study, we discovered functional polymer chemosensors that can exhibit ratiometric hydrostatic pressure signaling. The conceptual scheme, as depicted in Figure , involves the application of hydrostatic pressure to induce conformational changes in the polymer, resulting in perturbations in the fluorescence intensities of the monomer and excimer forms as ratiometric responses. The choice of pyrene as a fluorophore in the polymer design is crucial owing to its dual emissive behavior as a monomer and an excimer. For the dynamic polymer scaffold, we selected α-poly-L-lysine (α-PLL) and ε-poly-L-lysine (ε-PLL), with the difference in the number of methylene groups expected to play a significant role in the dynamics of the polymer backbones. The molecular design is greatly inspired by the fluorescence data of the pyrene-labeled PLLs and other flexible polymers under atmospheric conditions (0.1 MPa). Moreover, the degree of substitution (DS) of pyr on the polymer backbone is highly likely to significantly impact ratiometric signaling. Hence, we synthesized a series of varying DSs (low, middle, or high DS) of -PLLpyr and -PLLpyr conjugates, respectively, and pyrref as the reference monomer, as shown in Figure . Herein, we present the ratiometric responses observed upon hydrostatic pressurization using α-PLLpyr and ε-PLLpyr in different solvents, including MeOH, H2O, and PBS. The choice of PBS solution, which closely resembles the biological medium with an ionic strength of 166 mM, allows for potential applications of these polymer chemosensors in biocompatible materials. By conducting comparative studies in both hydrophilic organic solvents and aqueous solutions, we aim to gain deeper mechanistic insights into the factors influencing the ratiometric signaling behavior of the polymer chemosensors. These insights may eventually pave the way for applications in mechanobiological imaging. First, we investigated the ratiometric behaviors (the monomer/excimer ratio) of all the polymers at 0.1 MPa (atmospheric pressure). As shown in Figures and, both and -PLLpyrs showed a sudden increase in excimer emission at 500 nm with increasing DS in DMSO; naturally, the monomeric reference pyr-ref exhibited no excimer emission. This is attributed to the degree of crowding of pyr chromophores on the polymer backbones. Therefore, we will further investigate the effect of hydrostatic pressurization on these ratiometric responses, particularly in PBS. As a representative ratiometric polymer, -PLLpyrhigh in PBS (Figure ) was first examined. Importantly, under hydrostatic pressurization, the monomer emission intensity slightly decreased while the excimer intensity increased. This simple ratiometric response provides a means for detecting applied hydrostatic pressures. In contrast, as shown in Figures and in the SI, both α-PLLpyrlow and α-PLLpyrmid exhibited a mutual decrease in both monomer and excimer intensities with increasing hydrostatic pressure. The degrees of decrease varied in each polymer, resulting in inherent ratiometric characteristics, as shown in Table . Furthermore, the hydrostatic pressure-dependent fluorescence changes of α-PLLpyrs (and ε-PLLpyrs) in MeOH and H2O are presented in Figures S21-S24 in the SI. The observed changes in intensity ratios (monomer/excimer ratios) varied depending on the solvents and chromophore substitution (DSs) used, indicating that ratiometric signaling under hydrostatic pressurization can be controlled by various parameters.