Ascorbic acid (AA), dopamine (DA), uric acid(UA) and tryptophan(Trp) are considered as crucial small biomolecules for physiological processes in human metabolism. Abnormal levels of these species will lead to the occurrence of some diseases and disorders. In this paper, polyethyleneimine (PEI) covalent functionalized graphene(PEI-G) was used to modify glassy carbon electrode and applied for the simultaneous and sensitive determination of the four molecules. The PEI-G nanocomposite was characterized by fourier-transform infrared spectroscopy(FTIR), ultraviolet-visible (UV-Vis), X-ray powder diffraction(XRD) and transmission electron microscopy(TEM). The electrochemical behaviors of AA, DA, UA and Trp on PEI-G modified electrode were studied in detail. And the optimal experimental conditions for the simultaneous determination of AA, DA, UA and Trp were explored. The results showed that the oxidation peaks of the four species could be well separated and the peak to peak separations were 298 mV (AA-DA), 130 mV (DA-UA) and 350 mV(UA-Trp), respectively. The linear response ranges for the simultaneous detection of AA, DA, UA and Trp were 50-5800 mu mol/L, 30-2570 mu mol/L, 0.05-400 mu mol/L and 6-1000 mu mol/L, with the detection limits of 16.67 mu mol/L, 10 mu mol/L, 0.017 mu mol/L and 2 mu mol/L, respectively. The proposed method provides a promising strategy for the simultaneous detection of these species in practical application.