A two-step zero-length cross-linking procedure using active esters was successfully adopted for conjugating en-rofloxacin (EF) to human serum albumin (HSA). The derived conjugate was characterized by UV spectrum and then usedfor immunization of BALB/C mice. In enzyme-linked immunosorbent assay (ELISA) and competitive inhibition ELISA ex-periments, the derived antiserum exhibited high antibody titer (greater than 1: 250 000) as well as varied cross-reactivity(from 97.8% to 161.7%) to three analogs of EF belonging to fluoroquinolones family. But over the concentration rangestudied, no significant cross-reactivity was observed to other group of antibiotics (chloramphenicol, oxytetracycline, sul-phamethoxazole and nysfungin). It was confirmed that the synthesized immunogen was highly antigenic and elicited specificantibody responses in BALB/C mice against EF. A two-step zero-length cross-linking procedure using active esters was successfully adopted for conjugating en-rofloxacin (EF) to human serum albumin (HSA). The derived conjugate was characterized by UV spectrum and then used for immunization of BALB / C mice The enzyme-linked immunosorbent assay (ELISA) and competitive inhibition ELISA ex-periments, the derived antiserum exhibited high antibody titer (greater than 1: 250 000) as well as varied cross-reactivity (from 97.8% to 161.7%) to three analog of EF belonging to fluoroquinolones family. But over the concentration range staged, no significant cross-reactivity was observed to other group of antibiotics (chloramphenicol, oxytetracycline, sul-phamethoxazole and nysfungin). It was confirmed that the synthesized immunogen was highly antigenic and elicited specificantibody responses in BALB / C mice against EF.