A new in-situ photochemical kinetic fluorimetric method was proposed for the determination of biacetyl (BI). It is based on the sensitization of BI on the photochemical reaction of amaranth (AM). AM, a nonfluorescent compound was converted into an intensively fluorescent compound in a slightly alkaline medium by the sensitized photochemical reaction, and BI was indirectly determined by monitoring the change of the fluorescence intensity. The determination can be carried out by fixed-time method or tangent method. The kinetic behavior of the reaction and the effects of some experimental conditions were investigated and discussed. The calibration graph was rectilinear from 1.0 μg ml−1to 10.0 μg ml−1of BI (r= 0.999), the limit of detection was 1.0 ng ml−1, and the coefficient of variation was 0.44% for 0.90 μg ml−1of BI (n = 6). The mechanism for the sensitization of BI was examined and the triplet-triplet energy transfer, in which BI acted as the energy donor and AM as the energy acceptor, was suggested to be the main cause. Its application to real samples has been tested.