The management of fishery residues and plastics is considered to be a vital strategy for conserving resources and maintaining the quality of the environment. Poly(l-lactic acid) (PLA) is a commercially promising, renewable, and biodegradable plastic. In this study, a PLA depolymerase was produced in a squid pen powder (SPP) and recycled plastic waste (PLA powder)-containing medium by Pseudomonas tamsuii TKU015, a bacterial strain isolated from Taiwanese soil. This PLA depolymerase had a molecular weight of 58 kDa and was purified to homogeneity from the supernatant of a TKU015 culture. The optimum pH of TKU015 PLA depolymerase is 10, and the optimal temperature of the enzyme is 60 °C. In addition to PLA, TKU015 PLA depolymerase degraded fibrinogen and tributyrin, but did not hydrolyze casein, triolein, and poly(β-hydroxybutyrate). Taken together, these data demonstrate that P. tamsuii TKU015 produces a PLA depolymerase to utilize SPP and polylactide as carbon/nitrogen sources.