Fish bile is a biological fluid with great potential as a biomarker of environmental exposure to metals and complex mixtures. This study verified the potential of this matrix in environmental contamination situations by metals and analysed proteomic aspects. Two fish species (Mugil liza – mullets, and Tilapia rendalli – tilapias) were sampled from different locations. Tilapia individuals were exposed to sub-lethal Cu concentrations in the laboratory. Bile was analyzed regarding trace-element concentrations (Cd, Cu, Pb, Zn, Ni, Mn), and statistical techniques indicate that this matrix can be used in biomonitoring for metal exposure instead of liver, being more advantageous than the latter, since this is a less complex matrix, leading to easier pre-treatment, besides the possibility of not having to sacrifice the animal. Different clean-up protocols were tested with the aim of analysing bile by 1D and 2D electrophoresis and zymographies for protein and enzyme detection. The final clean-up protocol consists of sonication, centrifugation, delipidation and desalting. The presence of metallothioneins was analyzed in this matrix by 1D and 2D electrophoresis, spectrophotometry and SEC-HPLC-ICP-MS, and it was verified that there really is metallothionenin expression in bile, showing similar behaviour to that reported in liver, of increased expression in environmental contamination situations. We also verified the existence of matrix metalloproteases in bile by gelatinolinic zymographies, and these also showed potential as biomarkers for environmental contamination, since some gelatinolinic bands were present only in a contaminated site in comparison to a reference location. This study verified the existence of two protein biomarkers in fish bile, one specific to metal contamination (metallothioneins) and one non-specific (matrix metalloproteinases), besides the possibility of utilizing fish bile as a biomarker to metal exposure by the determination of trace-elements in this matrix.