The fabrication of stable fluorescent MoNCs (molybdenum nanoclusters) in aqueous media is quite challenging as it is not much explored yet. Herein, we report a facile and efficient strategy for fabricating MoNCs using 2,3 dialdehyde maltose‐cysteine Schiff base (DAM‐cysteine) as a ligand for detecting myoglobin and γ‐aminobutyric acid (GABA) in biofluids with high selectivity and sensitivity. The DAM‐cysteine‐MoNCs displayed fluorescence of bright blue color under a UV light at 365 nm with an emission peak at 444 nm after excitation at 370 nm. The synthesized DAM‐cysteine‐MoNCs were homogeneously distributed with a mean size of 2.01 ± 0.98 nm as confirmed by the high‐resolution transmission electron microscopy (HR‐TEM). Further, X‐ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT‐IR) techniques were utilized to confirm the elemental oxidation states and surface functional groups of the DAM‐cysteine‐MoNCs. After the addition of myoglobin and GABA, the emission peak of DAM‐cysteine‐MoNCs at 444 nm was significantly quenched. This resulted in the development of a quantitative assay for the detection of myoglobin (0.1–0.5 μM) and GABA (0.125–2.5 μM) with the lower limit of detection as 56.48 and 112.75 nM for myoglobin and GABA, respectively.