Objective: To explore the induced expression of human anti-Trop-2 antibody fragment Fab in E.coli TOP10F ′ and to optimize the expression and purification conditions of protein. Methods: The plasmid containing anti-Trop-2 Fab antibody gene was transformed into E.coli TOP10F ′. It was tested to replace the culture medium before induction of anti-Trop-2 Fab expression. The effects were compared in different induction temperatures,induction times and glucose added before induction on protein expression. Anti-Trop-2 Fab was purified by affinity chromatography and its immunological characteristics were evaluated by immunofluorescence and flow cytometry. Results: In the culture medium by adding 5 g/L of glucose,the expression of anti-Trop-2 Fab significantly increased. The temperature of 16℃ can induce more expression of Fab than other temperatures. The expression of anti-Trop-2 Fab reached peak after adding inducer for 12 hours. Conclusion: This study provides the basis of large-scale preparations of anti-Trop-2 Fab in prokaryotic expression system and subsequent utilization of anti-Trop-2 Fab in tumor targeted therapy.