L-Phosphinotricin (L-PPT) is an active compound of a broad-spectrum herbicide used worldwide. Asymmetric synthesis of L-PPT from 4-(hydroxy(methyl)phosphoryl)-2-oxobutanoic acid (PPO) using transaminase is of great significance due to the 100% theoretical yield and high enantioselectivity. In this study, a new transaminase from Pseudomonas fluorescens ZJB09-108 (named as Pf-TA) was cloned and expressed in E. coli BL21 (DE3). The amino acid sequence analysis showed that the maximum sequence identity of Pf-TA with other transaminases is 74%. This recombinant Pf-TA exhibited a good temperature stability and pH stability. The K-m and k(cat) of Pf-TA toward PPO were 34.02 mmol L-1 and 11.3 s(-1), respectively. After optimization of the L-PPT biosynthesis catalyzed by whole-cells of E. coli harboring transaminase, >99% yield of L-PPT with 99.9% of enantiomeric excess was attained at a PPO concentration of 100 mM. High yield of L-PPT (79%) with 99.9% enantiomeric excess was achieved at high PPO concentration of 500 mM as well. These results suggested the recombinant transaminase is a potential candidate for the biosynthesis of L-PPT from PPO.