Mycoplasma ovipneumoniae (MO) is main responsible for pleuropneumonia in sheep. The disease is spreading in the world, and has caused great suffering and losses to the sheep husbandry. Current methodologies for detecting and identifying MO are somehow time consuming, complicated, expensive. Herein, a powerful biosensing platform for effective MO detection has been explored based on manganese dioxide (MnO2) microsphere absorbing Cy5 labeled single strand DNA (Cy5-ssDNA) probe, in which different base number of the complementary of MO conserved sequence was employed. The results demonstrated that the fluorescence enhancement exhibited a good linear relationship with MO plasmid added over a concentration range of 10(2)-10(6) copies/mu L, with the detection limit of 1.042 copies/mu L. This biosensing platform has been finally employed as the feasible probe for MO plasmid and detection with good reproducibility and stability, which exhibited a good performance to discriminate perfect complementary target MO and the base mismatched DNA sequence, illustrated that using MnO2 microsphere absorbing Cy5-ssDNA probe as biosensor can detect MO in simple, rapid, economical, selective and sensitive way, and showed significantly potential in more extensive biochemical application. (C) 2017 Elsevier B.V. All rights reserved.