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[Display omitted] • Rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) from sterile body fluids. • Magnetic separation enriched bacteria and identified specific protein peaks by MALDI-TOF MS. • The enrichment efficiency in cerebrospinal fluid and blood was greater than 80.95 % and 83.6 %. • The whole detection process could be completed within 1.5 h. • It breaks through the problems of long time and high technical requirements of traditional bacterial culture. As a result of the extensive use of antibiotics, methicillin-resistant Staphylococcus aureus (MRSA) has emerged, leading to serious clinical and epidemiological problems. This study established a rapid and accurate method for detection of MRSA in clinical samples. The MRSA detection procedure involved a magnetic separation technique and Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the characteristic peaks of proteins were compared to achieve the detection of MRSA. We treated PBS, blood and cerebrospinal fluid with MRSA, and identified it after short-term culture. The time and detection limit for identifying MRSA were determined. At the same time, we used the method to detect different proportions of mixed bacteria. The results revealed that the enrichment efficiency of Amp-MBs for MRSA in PBS, blood and cerebrospinal fluid was greater than 95 %, 80.95 % and 83.6 %, respectively. The whole detection process could be completed within 1.5 h. The negative and positive percent agreement of MRSA detection were 83 % and 95 %, respectively. In mixed bacterial samples, bacteria can be detected when the proportion of MRSA is greater than 50 %. Overall, this study provides an effective method for bacterial preconcentration and detection, which has high potential for rapid and sensitive diagnosis of MRSA infection [ABSTRACT FROM AUTHOR]