Intrinsic milk photoluminescence (PL), though empirically observed, remains insufficiently explored in terms of mechanism and application. This work illustrates the general dual-emission characteristics of milk and elucidates their distinct origin: blue emission at 390–460 nm from casein and whey protein aggregates via clustering-triggered emission and yellow-green emission at around 530 nm from riboflavin. Crucially, microbial metabolism during spoilage induces pronounced physicochemical transformations: lactic acid accumulation that drops the pH from 6.69 to 4.79 within 72 h, extensive protein degradation with a 200-fold increase in free proline, and colloidal reorganization from uniform particles to polydisperse aggregates. These changes dynamically modulate PL signatures: early-stage (<12 h) riboflavin decay induces blueshifted emission, while advanced spoilage (24–72 h) disrupts protein aggregation, reducing quantum yield