Objective To evaluate the value of next-generation sequencing (NGS) technique in detecting potential pathogens in valves of patients with infective endocarditis (IE), and provide help for the diagnosis and postoperative treatment of IE. Methods According to modified Duke criteria, valve tissues of IE patients and patients who were excluded IE were performed culture conventionally,pathogens in valve tissues were detected directly by NGS technique, results were compared with blood culture and pathological smear staining. Results The sensitivity, specificity, positive predictive value, and negative predictive value of NGS were 95.0%, 85.7%, 95.0%, and 85.7% respectively, blood culture were 30.0%, 100.0%, 100.0%, and 30.0% respectively, valve culture were 10.0%, 100.0%, 100.0%, and 28.0% respectively. Conclusion Compared with conventional culture method, NGS technique has higher sensitivity and shorter time in detecting IE valve vegetation. NGS is of great value in the diagnosis and treatment of IE, especially in suspected IE with negative culture results.