A method is reported for the synthesis of highly luminescent copper/molybdenum bimetallic nanoclusters (Cu/Mo NCs) using cysteine as both a capping and reducing agent. The nanoclusters display bluish-green luminescence (excitation/emission peaks at 370/490 nm) and a relative quantum yield of 26%. The capped Cu/Mo NCs were used as a fluorescent probe for determination of the antineoplastic drug methotrexate (MTX) via an inner filter effect. Fluorescence intensity decreases linearly in the 50 nM to 100 μM MTX concentration range. The limit of detection is 13.7 nM. This approach has been successfully applied to the determination of MTX in spiked human urine with a typical recovery of 99%.