【Objective】 Phenylalanine ammonia-lyase (PAL) plays an important role in the phenylpropanoid metabolism pathway. It would be of importance to determine the PAL genes from Populus trichocarpa, analyze their enzymatic properties, and apply them in biosynthesis. This study provides a reference for the research of its phenylpropanoid metabolic pathway and synthesis of trans-cinnamic acid (t-ca). 【Method】 Based on the genomic annotation information, the PtrPAL genes of P. trichocarpa were searched. Some of them were selected for prokaryotic expression and enzymatic analysis. Finally, t-ca was biosynthesized from L-phenylalanine (L-Phe) by whole-cell catalysis. 【Result】 In this paper, fivePAL genes were predicted in the genome of P. trichocarpa. They were divided into two groups based on the phylogenetic tree analysis. The PtrPAL3 and PtrPAL4, which from different groups, were selected for protein expression in Escherichia coli and enzymatic analysis. The optimum temperature of rePtrPAL3 and rePtrPAL4 was 55 °C and 60 °C, respectively. The optimum pH was 8.5 and 8.0, respectively. The rePtrPAL3 (3.363 U/mg) and rePtrPAL4 (3.381 U/mg) exhibited similar PAL activity and weak tyrosine ammonia-lyase activity. Kinetic analysis indicated that rePtrPAL3 has higher affinity for L-Phe; therefore, it was used to produce t-ca from L-Phe, and the titer reached 104 mmol/L in 7 h by whole-cell catalysis. 【Conclusion】 In spite of different transcriptional specificities, the two PtrPALs displayed similar enzymatic properties. However, rePtrPAL3 showed higher catalytic efficiency. Thus, rePtrPAL3 is more suitable for the biosynthesis of t-ca. In addition, it has good application potential for that the titer of t-ca is higher than other reports (28 mmol/L).