Identification of body fluids from forensic biological samples is an essential procedure in a criminal investigation. In this study, a systematic method using ELISA for the detection of statherin (STATH), dermcidin (DCD) and Tamm-Horsfall protein (THP) has been developed to identify saliva, sweat and urine, respectively. In addition, it was examined whether this method is suitable for use on body fluid stains, aged stains, and simulated casework samples. Furthermore, the sensitivities of ELISA for STATH, DCD and THP were compared with those of presumptive tests for saliva, sweat and urine, respectively. As a result, saliva, sweat and urine were successfully identified by using the systematic method of ELISA developed in this study. STATH was successfully detected in 2.5-year-old saliva stains and simulated casework samples such as a cigarette butt. Limit of detection (LOD) of STATH by ELISA equated to 31.3 nl of saliva. It would be enough to apply to casework samples although higher than that of blue starch-agarose test. DCD was successfully detected in 7-year-old sweat stains and simulated casework samples such as the surface of a cellular phone. LOD of DCD by ELISA equated to 31.3 nl of sweat, and it was almost similar to that of the Determiner-LA test. THP was successfully detected in 4-year-old urine stains. LOD of THP by ELISA equated to 9.8 nl of urine, and it was almost similar to those of presumptive tests for urea and uric acid. In conclusion, ELISA for the detection of STATH, DCD and THP could be an effective tool for the forensic identification of saliva, sweat and urine because of its specificity and sensitivity. ELISA for the detection of STATH, DCD and THP should be applied to supplement presumptive tests for saliva, sweat and urine.