In this study, a series of biodegradable polyurethane films (PU-Fs) is prepared by step-growth polymerization of hydroxylated soybean oil (SBO-OH) and L-lysine diisocyanate ethyl ester (L-LDI) that are renewable-based di-functional building blocks. The influence of loading ratio by weight (SBO-OH/L-LDI (w/w) = 1:0.5; 1:1 and 1:1.5) on the biodegradable, mechanical and thermal properties of final PU-Fs is systematically investigated. In the first step, epoxy groups of epoxidized soybean oil are converted to corresponding hydroxyl functionalities to react with the diisocyanate groups of L-LDI via film casting method at room temperature. The obtained PU-Fs with higher L-LDI loading exhibit higher thermal and mechanical properties as well as more hydrophobic characteristic compared to others. Moreover, biodegradability of resulting PU-Fs is also studied using hydrolytic and enzymatic degradation experiments. Noteworthy, it is found that around 50 and 60% of PU-Fs are degraded by enzymatic and hydrolytic experiments after 12 weeks.