Abstract The electrochemical behavior of chloramphenicol (CAP) at a graphite electrode has been investigated by parallel incident spectroelectrochemistry (PISEC) accompanied by cyclic voltammetry. The redox electrode reaction mechanism of CAP under different pH conditions has been described. In NH 4Cl–NH 4OH buffer solution (pH 8.0), CAP undergoes an irreversible 4-electron reduction at negative potential to give NHOH-CAP. Consequently, a new redox-active species is formed at more positive potentials, which has been identified as the NHOH-CAP/NO-CAP couple. In 0.1 mol/l HCl media, CAP undergoes an irreversible 6-electron transfer, corresponding to reduction of the nitro group to amine. The content of CAP in eyedrops has been determined by PISEC in ammonia buffer solution (pH 8.0) under the initial reduction of CAP. The linear range of quantitation was 1.0×10 −6 mol/l∼2.5×10 −4 mol/l, with a correlation coefficient of 0.999. The determination recoveries were 95.5%∼101.5%.