Abstract: Objective To study the clinical distribution and drug resistance isolated from cerebrospinal fluid and other sterile body fluid (pleural effusion, ascites, bile, etc.) in our hospital from 2012 to 2014, and provide guidance for clinicians in the rational use of antibiotic. Methods The isolation rate, distribution and drug resistance of the first isolated strains isolated from cerebrospinal fluid and other sterile body fluids (such as pleural effusion, ascites, bile) in our hospital from 2012 to 2014 were retrospectively analyzed. Results From 2012 to 2014, 120 strains, 141 strains, 160 strains of sterile body fluid specimens were separated, respectively. The most common specimen was gram-negative bacilli with separation rate of more than 70%, followed by gram-positive bacteria and fungi, with the separation rate of more than 35% and 8%, respectively. Prostate fluid, bile, cerebrospinal fluid's separation rate increased year by year, which were all more than 30%. From prostate fluid specimens, we mainly isolated positive coccus, in which epidermis staphylococcus was the most; From bile and chest water, we mainly isolated negative bacilli, in which E.coli was the most. The resistance rate of epidermis staphylococcus and hemolytic staphylococci to clindamycin and erythromycin were more than 60%; Penicillin's resistance rate was more than 85%; No strains were found to be resistant to Nitrofurantoin, Quinupristin/dalfopristin, Tigecycline, Vancomycin and Teicoplanin in 3 years. The resistance rate of E. coli to quinolone was more than 60%, with good sensitivity to Piperacillin/tazobactam, Cefotetan, while it had no resistance to Imipenem, Amikacin, Nitrofurantoin and Meropenem. The sensitivity of Klebsiella pneumoniae was less than E. coli to Meropenem, Nitrofurantoin, Cefotetan and Imipenem.The resistance rate of Klebsiella pneumoniae (CRE) in Hydrocarbon mildew resistant enzyme increased gradually in three years and reached even more than 15%. Pseudomonas aeruginosa was more sensitive to Amikacin; but it had severe drug-resistant to Ampicillin/sulbactam, Cefotetan, Ceftriaxone, Cefazolin, Cefuroxime sodium, SMZ-TMP, Nitrofurantoin and Cefuroxime Axetil, with the average drug resistance rate of more than 90%. Conclusion The resistant strains of pathogenic bacteria in sterile body fluids show a rising trend. Klebsiella pneumoniae is found to be resistant to carbon penicillium enzyme. It is important for clinicians to monitor the changes of pathogenic bacterial flora and their drug resistance tendency promptly, so as to decrease the production of drug-resistant strains.