The present study aims to investigate the physicochemical characteristics of phenylalanine ammonia-lyase (PAL) extracted from agricultural waste and its potential use as an anticancer agent in comparison to microbial PAL. We extracted and partially purified PAL from agricultural waste sources. We assessed the temperature and pH range of PAL and determined enzyme kinetics parameters including Michaelis constants (Km), maximum velocity (Vmax), and specificity constant values (Vmax/Km). Additionally, we examined the effects of different storage temperatures on PAL activity. In our analysis, we compared the efficacy of agricultural waste-derived PAL with PAL from Rhodotorula glutinis. The results demonstrated that PAL extracted from agricultural waste exhibited significantly higher specific activity (Vmax/Km) compared to its microbial counterpart. The agricultural waste-derived PAL displayed a stronger affinity for phenylalanine, as indicated by a lower Km value than the microbial PAL did. Furthermore, PAL from agricultural waste maintained activity across a broader temperature and pH range (15–75 °C, pH 5–11), in contrast to microbial PAL (20–60 °C, pH 5.5–10). Importantly, the PAL derived from agricultural waste exhibited superior stability, retaining over 90% of its activity after 6 months of storage at room temperature (25 °C), whereas microbial PAL lost more than 70% of its activity under similar storage conditions. In anticancer experiments against various cancer cell lines, agricultural waste-derived PAL demonstrated greater anticancer activity compared to microbial PAL. These findings suggest that PAL sourced from agricultural waste has the potential to be a safe and effective natural anticancer agent.