An immunoglobulin Y (IgY) based indirect double antibody sandwich enzyme linked immunosorbent assay (ELISA) was developed for the detection of Indian cobra (Naja naja naja) venom in the biological samples of forensic origin. Polyclonal antibodies were raised and purified from chick egg yolk and rabbit serum. The cobra venom was sandwiched between immobilized affinity purified IgY and the rabbit IgG. The detection concentration of cobra venom was in the range of 0.1 to 300 ng. The calibration plot was based on linear regression analysis (y = 0.2581x + 0.4375, r2 = 0.9886). The limit of detection of the assay was found to be 0.1 ng. The coefficient of variation (CV) of different concentrations of working range in inter (n=6) and intra-assay (n=6) was observed to be less than 10%. The recovery of venom was found to be in the range of 80–99%, when different concentrations (0.002, 0.1, 0.2, 1, and 2 μg) of cobra venom were spiked to pooled normal human serum (ml−1). No cross reactivity was observed with krait and viper venom in the immunoassay system in the concentration range of 0.1–1000 ng. The method was initially, validated by analyzing specimens (autopsy) of experimental rats injected with cobra venom (1.2 mg kg−1 body mass). Further, human specimens (autopsy and biopsy) of snake bite victims of forensic origin were also analyzed. The methodology developed may find diagnostic application in forensic laboratories.