The usefulness of fish biomarkers for freshwater biomonitoring is now well recognized, but they still pose several questions to ecotoxicology researchers. The present study, designed to assess the effects of a small city located in an agricultural river basin watershed on sticklebacks living in an adjacent river, underlines the importance of reference selection. Two reference systems were used to analyse responses of a set of biomarkers, including biotransformation enzymes, oxidative stress parameters, neurotoxicity and endocrine disruption end-points, measured in wild sticklebacks electrofished in a contaminated stream. The results showed that the investigated urban pressure disturbed CYP3A activity but also induced hepatic lipoperoxidation and circulating vitellogenine but this result is strongly influenced by the selected reference system. This work therefore demonstrates the need for further research to identify a robust reference system for stickleback biomarker analysis.