The introduction of fluorinated amino acid carrying a CF3 moiety in therapeutical agents and protein engineering requires the accessibility of highly pure chiral samples in order to correctly understand the effect of fluorination on bioactivity. Here we report an easy enzymatic approach for the synthesis of (2S)-5,5,5-trifluoroleucine and its subsequent resolution into its (2S,4S) and (2S,4R) diastereoisomers. This strategy stands for a sustainable synthesis and enhanced resolution by means of alternative protecting groups for the amino/acid functionalities than that previously reported.