An effective biotin-avidin amplified enzyme-linked immunosorbent assay (BA-ELISA) was developed to determine ketamine in biological samples. A conjugate of ketamine and ovalbumin (OVA) was used for immunization to produce polyclonal antibody. The conjugate of ketamine and bovine serum albumin (BSA) with polyclonal antibody was calculated to have an affinity constant (K(aff)) of 3.30 x 10(8)(mol/L)(-1). The linear range of ketamine was 0.1-1000 microg/L with recoveries from 89.6% to 99.9% in spiked sample analysis. The detection limit of ketamine was 0.03 microg/L, which is more sensitive than that of the traditional ELISA. The results obtained by BA-ELISA agreed well with that of the traditional ELISA, with a correlation coefficient of 0.98.