【Abstract】Purposes Bloodstream infection (BSI) has a high mortality and disability rate, and accurate and timely pathogen diagnosis is crucial. Our aim is to evaluate the value of droplet digital PCR (ddPCR) in diagnosing bloodstream infections. Methods We conducted a prospective study, collecting 55 patients suspected of bloodstream infection and comparing droplet digital PCR detection (approximately 3 hours) with traditional blood culture (BC, at least 48 hours). Results The sensitivity and negative predictive value of ddPCR were high, and the positive rate was significantly higher than that of blood culture; It was not significantly correlated with the inflammatory factors procalcitonin (PCT), C-reactive protein (CRP), and white blood cells (WBC) in plasma; However, in the ddPCR+/BC+ group, the number of gene copy of pathogenic bacteria detected by ddPCR showed a synchronous trend with PCT, CRP, and WBC. In addition, all the six samples with carbapenem-resistant Klebsiella pneumoniae isolates identified through antimicrobial susceptibility testing were also detected by ddPCR, with serinase (blaKPC) or metalloenzymes (blaNDM and blaIMP). The mecA gene was also detected in all the three samples with methicillin-resistant Staphylococcus isolates. Moreover, ddPCR detected antimicrobial resistance genes in other nine culture-negative samples. Conclusion Droplet digital PCR is a rapid pathogen-detecting method that can be supplementary to traditional blood culture. When combined with clinical symptoms, ddPCR can be used to quickly diagnose patients with suspected bloodstream infections. At the same time, it also has potential advantages in detecting antimicrobial resistance genes.