The production of L-DOPA using L-tyrosine as substrate, the enzyme tyrosinase (EC 1.14.18.1) as biocatalyst, and L-ascorbate as reducing agent for the o-quinones produced by the enzymatic oxidation of the substrates was studied. Tyrosinase immobilization was investigated on different supports and chemical agents: chitin flakes activated with hexamethylenediamine and glutaraldehyde as crosslinking agent, chitosan gel beads, chitosan gel beads in the presence of glutaraldehyde, chitosan gel beads in the presence of poly-vinylpyrrolidone, and chitosan flakes using glutaraldehyde as crosslinking agent. The last support was considered the best using as performance indexes the following set of immobilization parameters: efficiency (90.52%), yield (11.65%), retention (12.87%), and instability factor (0.00). The conditions of immobilization on chitosan flakes were optimized using a two-level full factorial experimental design. The independent variables were enzyme-support contact time (t), glutaraldehyde concentration (G), and the amount of enzyme units initially offered (U-C). The response variable was the total units of enzymatic activity shown by the immobilized enzyme (U-IMO). The optimal conditions were t = 24 h, G = 2% (v/v), and U-C = 163.7 U. Under these conditions the total units of enzymatic activity shown by the immobilized enzyme (U-IMO) was 23.3 U and the rate of L-DOPA production rate was 53.97 mg/(L.h).