Prussian Blue nanocubes / carbon nanospheres (PB-CNS) heterostructure composed of perfectly cube and spherical composite on indium tin oxide (ITO) surface were developed for in vitro sensing of anti-diabetic drug i.e. Sitagliptin (STA). Detailed morphological, electrochemical, structural and optical characterization of PB-CNS/ITO electrode was done using DLS, SEM, EIS, CV. The sensor showed rapid response time (within 5 s) and the linear range from 0.001 to 10 mM with a shelf life of about 10 weeks under refrigerated conditions. We have also attempted to employ this electrode for assessment of STA in urine samples. The developed sensor exhibited high reproducibility and good storage stability.