Plasmin play an important role in milk spoilage and can cause quality issues in ultra-heat treated (UHT) milk and other dairy products. Current methods for measuring plasmin activity in milk have low sensitivity and are too slow to be used in routine testing. We report a bioluminescence resonance energy transfer (BRET)-based biosensor that can measure naturally occurring low levels of plasmin activity in milk within a few minutes. The biosensor incorporates a plasmin-specific peptide target sequence flanked by the resonance energy BRET transduction elements: green fluorescent protein (GFP2) at the N-terminus and a variant Renilla luciferase (RLuc2) at the C-terminus. Complete cleavage of the peptide linker sequence by human plasmin led to an approximately 87% decrease in the starting BRET ratio. Using a 10 min incubation time, the detection limit for human plasmin was 0.25 nM and 0.86 nM for bovine plasmin in 50% (v/v) full fat milk with EC50‘s of 5.89 ± 0.12 nM and 5.97 ± 0.59 nM, respectively. These detection limits are below the naturally occurring levels of plasmin reported to occur in raw or processed cow’s milk. The plasmin biosensor therefore has the potential to measure naturally occurring plasmin levels directly in raw and UHT-processed milk samples. The protease biosensor described herein is selective and sensitive and retains its function in the complex colloidal matrix of milk. It therefore meets many of the requirements for routine use in production settings. We propose its use for managing plasmin-related quality issues in UHT milk and other dairy products.