Objective To establish a rapid and accurate method of bacterial identification in order to shorten turnaround time of positive blood cultures. Methods A total of 91 bacteria were collected from positive blood cultures, incubated in nutrient broth and blood agar plate respectively for 4 h and then identified by matrix-assisted laserdesorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). Taking the results of MALDI-TOF MS of 24-h blood plate culture as the standard, the coincidence rates of the two incubation methods for identification of bacteria by mass spectrometry were compared. Results Among the 91 bacteria there were 51 strains of Gramnegative bacteria and 40 strains of Gram-positive bacteria. In all, 46.15% and 89.01% of the isolates were accurately identified after 4-h incubation in nutrient broth and blood plate respectively, while there were 47.25% and 3.30% strains with no results, and the error identification rates were 3.30% and 4.40%, respectively. The agreement rates of the two methods had significant difference (P < 0.05). The coincidence rates of 4-h incubation in nutrient broth and blood plate were 58.82% and 94.12% respectively for Gram-negative bacteria at species level (P < 0.05), the latter was significantly higher than the former. The coincidence rates of 4-h incubation in nutrient broth and blood plate were 30.00% and 82.50% for Gram-positive bacteria (P < 0.05), the accuracy of blood agar plate inoculation was better than that of nutrient broth culture. Conclusions Using technology of MALDI-TOF MS, rapid bacteria identification of positive blood culture samples can be achieved after 4-h incubation in blood agar plate.