This manuscript presents in-situ electrochemical synthesis of Prussian Blue-gold nanoparticles (PB- AuNPs) composite for application in hydrogen peroxide (H2O2) biosensor. The SEM image clearly showed the presence of AuNPs of size in range of 50 to 200 nm spread on PB matrix. UV-Visible spectra showed absorbance peak at 530 nm corresponding to AuNPs and a hump in 690-740 nm region for PB, confirming the synthesis of composite. The cyclic voltammetry (CV) showed the surface coverage of 3.65 x 10 -9 mol/cm 2 for pure PB film and 4.33 x 10 -9 mol/cm 2 for PB-AuNPs film, with diffusion coefficient of 1.19 x 10 -9 cm 2 /s, and 5.64 x 10 -9 cm 2 /s respectively. The film thickness is found to be 2.4 x 10 -12 cm for PB and 2.9 x 10 -12 cm for PB-AuNPs composite. The concentration of redox active centers (Fe +3/+2 ) is 3.5 moles/cm 3 for ITO/PB and 4.1 moles/cm 3 for ITO/PB-AuNPs respectively. The CV of ITO/PB showed one redox couple at 0.118 V and 0.215 V, whereas with ITO/PB-AuNPs electrode, two sets of well-defined redox peaks; (i) 0.095 V & 0.135 V and (ii) 0.74 V & 0.78 V were obtained. The faradic current obtained with ITO/PB was 3.6 x 10 -3 A and 7.3 x 10 -3 A for ITO/PB-AuNPs composite film, respectively. The faradic current was almost double in presence of gold nanoparticles, as compared to pure PB. For H2O2 biosensor, the horse radish peroxidase (HRP) was immobilized on composite film and was used for H2O2 detection. The linearity was obtained from 10 to 90 nM, with sensitivity of 0.73µA/nM and the apparent Km value was 45 nM. The response time of reported biosensor is 20 sec and is stable for about three months.