The invention provides a penicillin G acylase mutant for synthesis and an application thereof in the preparation of amoxicillin. Penicillin G acylase of Achromobacter xylosoxidans origin is mutated by computer aided design in connection with semi-rational design of site-saturation mutagenesis technique and enzyme engineering modification of orthogenesis, thus acquiring the penicillin G acylase mutant lower in hydrolytic activity, better in synthetic activity, higher in synthesis-hydrolysis ratio (S/H), higher in acid resistance and better in stability, and amoxicillin can be catalytically synthesized more effectively and quickly. Immobilized enzyme hydrolytic activity of the mutant SPGA-4 obtained is decreased by 8.7 times, synthetic activity is increased by 5.6 times, the S/H ratio is increased by 8 times, the activity remains at 79% for 60 min under the condition of pH 2.0, amoxicillin is catalytically synthesized by a solid method at 10 DEG C or 20 DEG C, substrates 6-APA and D-HPM are directly charged in a solid form without dissolving, reaction pH need not be controlled, substrate conversion rate is higher than 99%, continuous use is available in more than 300 batches, and good operational stability is given.