The Middle Eocene Senj Mo–Cu deposit (1.3 Mt at 1.5 wt % Cu and 0.2 wt % Mo) is located in the central Alborz magmatic belt, northern Iran. Mineralization is characterized by multistage veins, which are hosted in volcanic and volcano-sedimentary strata of the Karaj Formation (c. 41–45 Ma), genetically associated with the Senj Mafic Sill (c. 40 Ma). Four generations of veins are documented based on mineral assemblages and cross-cutting relationships, sequentially: quartz–biotite–chalcopyrite (QBC veins), quartz–molybdenum (QM veins), quartz–pyrite (QP veins) and late calcite (LC veins). Three types of fluid inclusions (FIs) are present in multistage veins: liquid-rich two-phase (L-type), vapour-rich two-phase (V-type) and solid-bearing multi-phase (S-type) inclusions. FIs in the QBC and QM veins are predominantly V-type and S-type, together with minor L-type, whereas the QP and LC vein minerals only contain L-type fluid inclusions. The homogenization temperatures of FIs from QBC to LC veins vary from 320 to 425 °C, 308 to 390 °C, 214 to 288 °C and 145 to 243 °C, and their salinities range from 5.3 to 49.0, 6.2 to 39.7, 5.7 to 13.0, and 2.1 to 7.2 wt % NaCl equiv., respectively. The H–O–C isotope compositions favour a dominantly magmatic origin for the hydrothermal fluids, which were gradually diluted by meteoric waters. The S–Pb isotope data of sulphide minerals indicate that the sulphur and metals were sourced from a deep-seated magmatic reservoir. The Senj Mafic Sill and associated Mo–Cu mineralization was developed in a back-arc extension regime, which faults and crustal fracture systems served as conduits for hydrothermal fluid circulation and Mo-rich veining.