A novel and simple methodology for the detection of phosphinothricin produced by a biosynthesis approach in 96-well microtiter plates was developed based on the fluorometric determination of an isoindole derivative. The assay method to determine the generation of L-phosphinothricin was conducted with the help of a derivatization reaction. The linear detection range of the method was demonstrated to be from 0.74 to 100 mu g ml(-1) for the catalysis by resting cells and from 1.6 to 100 mu g ml(-1) for the catalysis by secretory enzymes. Meanwhile, the value of the relative standard deviation was less than 2.2% and the recovery was 99-101% of true value. Based on the method constructed in this study, one bacterial strain, Kluyvera cryocrescens ZJB-17005, with high stereo selectivity (> 99%) and excellent yield (45%) was isolated from 13,284 strains in the production of L-phosphinothricin by a biosynthesis approach.