In this contribution, bovine serum albumin stabilized gold nanoclusters as novel fluorescent probes were successfully utilized for the detection of methotrexate for the first time. Our prepared gold nanoclusters exhibited strong emission with peak maximum at 633.5 nm. However, the addition of methotrexate induced the strong fluorescence intensity of the gold nanoclusters to decrease. The decrease in fluorescence intensity of the gold nanoclusters caused by methotrexate allowed the sensitive detection of methotrexate in the range of 0.0016 μg mL⁻¹ to 24 μg mL⁻¹. The detection limit for methotrexate is 0.9 ng mL⁻¹ at a signal-to-noise ratio of 3. The present sensor for methotrexate detection possessed a low detection limit and wide linear range. In addition, the real samples were analyzed with satisfactory results.