To trace the most concerned bioavailable mercury (Hg) in aquatic environment, fish samples were collected from three typical regions in China, including 3 rivers and 1 lake in the Tibetan Plateau (TP, a high altitude background region with strong solar radiation), the Three Gorges Reservoir (TGR, the largest artificial freshwater reservoir in China), and the Chinese Bohai Sea (CBS, a heavily human-impacted semi-enclosed sea). The Hg isotopic compositions in fish muscles were analyzed. The results showed that anthropogenic emissions were the main sources of Hg in fish from TGR and CBS because of the observed negative δ²⁰²Hg and positive Δ¹⁹⁹Hg in these two regions (TGR, δ²⁰²Hg: − 0.72 to − 0.29‰, Δ¹⁹⁹Hg: 0.15 – 0.52‰; CBS, δ²⁰²Hg: − 2.09 to − 0.86‰, Δ¹⁹⁹Hg: 0.07 – 0.52‰). The relatively higher δ²⁰²Hg and Δ¹⁹⁹Hg (δ²⁰²Hg: − 0.37 – 0.08‰, Δ¹⁹⁹Hg: 0.50 – 1.89‰) in fish from TP suggested the insignificant disturbance from local anthropogenic activities. The larger slopes of Δ¹⁹⁹Hg/Δ²⁰¹Hg in fish from TGR (1.29 ± 0.14, 1 SD) and TP (1.25 ± 0.06, 1 SD) indicated methylmercury (MeHg) was produced and photo-reduced in the water column before incorporation into the fish. In contrast, the photoreduction of Hg²⁺ was the main process in CBS (slope of Δ¹⁹⁹Hg/Δ²⁰¹Hg: 1.06 ± 0.06, 1 SD). According to the fingerprint data of Hg isotopes, the most important source for aquatic bioavailable Hg in TP should be the long-range transported Hg, contrasting to the anthropogenic originated MeHg from surface sediments and runoffs in TGR and inorganic Hg from continental inputs in CBS. Therefore, the isotopic signatures of Hg in fish can provide novel clues in tracing sources and behaviors of bioavailable Hg in aquatic systems, which are critical for further understanding the biogeochemical cycling of Hg.