Background: Bloodstream infection (BSI) has been one of the biggest headaches for clinicians, as it not only aggravates symptoms but also increases the length of stay, the cost of hospitalization, and the side effects caused by antibiotics. It is an urgent need for clinicians to develop timely and accurate methods to find microorganisms. Currently, the gold standard for diagnosing BSI is blood culture, but it takes three to eight days to produce results, and its positive rate is extremely low. Next-generation sequencing (NGS) has emerged as a better technology desperately needed by doctors and patients to diagnose BSI. Objectives: This study compared NGS and blood culture methods in clinical patients with BSI. Methods: In this study, blood culture and NGS were used to analyze the blood of patients with BSI in different departments of the First Affiliated Hospital of Kunming Medical University. Results: Next-generation sequencing detected 60 pathogens in 63 blood samples, while blood culture detected 15 pathogens in 336 blood samples from 63 patients who were clinically considered to be infected. Pathogens detected by NGS included bacteria, fungi, and viruses, while blood culture only found bacteria and fungi. The positive rates of blood culture diagnosis and NGS diagnosis in BSI patients were 23.8% (15/63) (CI: 13.3%-34.3%) and 95% (60/63) (CI: 90%-100%), respectively. Conclusions: Our results showed that NGS creates a new diagnostic platform for patients with BSI. Its wide detection range, high positive rate, and characteristics of rapid detection will benefit patients with BSI. © 2020, Author(s).