Background BioCore recently developed a third generation of HIV 1/2 ELISA 3.0 kit using recombinant antigen of HIV1(gp41, p24) and HIV2(gp36). The kit detects HIV1/2 simultaneously and we evaluated the kit to validate it for diagnostic use. Methods We ran 200 negative samples and 100 positive samples using Green Cross HIV ELISA and BioCore kit to study the corelation between methods. PR16 serial dilution samples were used to test sensitivity and mixed serum and plasma samples were used to test sample matrix effect. Also the BBI seroconversion panel test was performed to compare performance of the kits; Anti-HIV 1/2 Combo performance panel(PRZ2 05 : 15 samples), Anti-HIV 1 seroconversion Panel X(PRB 924 : 8 samples), HIV P24 antigen sensitivity panel(PRA 801 : 10 samples),Anti-HIV Low titer performance panel(PRB 107M : 15 samples), Worldwide HIV performance Panel(WWRB 303 : 15 samples), Anti-HIV 1 seroconversion Panel Q(PRB 917 : 7 samples), Anti-HIV 1 seroconversion panel J(PRB 910 : 7 samples). Finally, 100 positive BioCore ELISA samples were confirmed by Western blot. Results The consistancy of 300 sample results was 100% for both Green Cross and BioCore ELISA. Also the detection limit of both kits was the same as 32 times dilution of PR 16 sample. There was no sample matrix effect of serum and plasma and BBI seroconversion panel test results were identical for both kits. Western blot results of 100 BioCore ELISA positive samples showed all positive. To determine kit stability, we performed stability tests for 13 months and we concluded BioCore ELISA kit was stable for 12 months from manufacturing date. Conclusion BioCore ELISA kit results of 7 BBI seroconversion panels were identical to the specifications and both sensitivity and specificity were excellent, respectively 100%. Also BioCore ELISA kits are stable up to 12 months from manufacture date. So it is a very useful screening kit to be used cost effectively for hospitals.