A novel glucose biosensor was designed using cobalt oxide (Co₃O₄) nanosheets patterned by π-conjugated polyaniline nanofibers (PANINFs). A facile synthesis process was conducted to obtain cost-effective and ecofriendly mesoporous Co₃O₄@PANINFs hybrid nanomaterial for the first time. The Co₃O₄@PANINFs on glassy carbon electrode (GCE), working as a biosensor electrode based on electrochemical technique, showed electrocatalytic activity to glucose with sensitivity of 14.25 μA mM^?1 cm^?2, linear range from (0.1 to 8) mM, minimum detection limit of 0.06 mM, and response time <6 s. Moreover, the biosensor was employed to monitor glucose concentration in human serum sample to provide effective sensing results.