Two novel impedimetric biosensors for highly sensitive and rapid quantitative detection of diazinon in aqueous medium were developed using two types of lipase, from Candida Rugosa (microbial source) (CRL) and from porcine pancreas (animal source) (PPL) immobilized on functionalized gold electrode. Lipase is characterized to specifically catalyze the hydrolysis of ester functions leading to the transformation of diazinon into diethyl phosphorothioic acid (DETP) and 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMHP). The developed biosensors both presented a wide range of linearity up to 50 M with a detection limit of 10 nM for Candida Rugosa biosensor and 0.1 RM for porcine pancreas biosensor. A comparative study was carried out between the two biosensors and results showed higher efficiency of Candida Rugosa sensor. Moreover, it presented good accuracy and reproducibility, had very good storage and multiple use stability for 25 days when stored at 4 degrees C.