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[Display omitted] • The effect of FLX on ES alkaline dark fermentation was explored for the first time. • FLX over 0.6 mg/Kg inhibited every step associated with biohydrogen production. • High FLX inhibited the release of nutrients from ES anaerobic fermentation. • High content of FLX reduced the activities of key enzymes related to biohydrogen production. Recently, the influence behavior of new pollutants in the environment has been widely concerned. However, the effect of antidepressants widely detected in excess sludge (ES) on biohydrogen production from anaerobic dark fermentation has never been explored. To fill this gap, fluoxetine (FLX), a typical antidepressant, was selected to evaluate its effect on ES mesophilic anaerobic dark biohydrogen fermentation. The results showed that FLX reduced biohydrogen production even at low content (0.1 mg/Kg). The biohydrogen yield was only 12.8 mL/g in the 1.8 mg/Kg (based on total suspended solids) FLX group, decreased by about 34.7%, compared with the control group (without FLX). Further mechanism investigation implied that high levels (more than 0.6 mg/Kg) of FLX reduced every step associated with the biohydrogen production. FLX reduced the concentration of ammonia nitrogen and phosphate in fermentation broth. FLX also had a significant negative effect on enzyme activity in ES dark fermentation. [ABSTRACT FROM AUTHOR]