Purpose: To evaluate the effects of blood culture’s clinical practice guideline on nurse’s knowledge, cognition, practice and false positive rates. Methods: Research instrument was developed to assess knowledge, cognition, practice of nurses regarding blood culture by investigators. It consists of 26 questions and verified validity. One hundred-two nurses participated in this study. A descriptive analysis was conducted to determine the subject’s characteristics and t-test, and Pearson’s correlation were conducted to evaluate the effects of guideline using SPSS 21.0. Results: After educating this guideline, nurse’s knowledge, cognition and practice of nurse significantly improved before education. The relations of correlation among knowledge and cognition, practice and knowledge, practice and cognition were statistically significant. In addition, although it was not statistically significant, false positive rate was decreased (pre-education 1.5%, post education 1%). Conclusion: The clinical practice guideline of blood culture may improve the nurse’s knowledge and nursing practice to decrease false positive rate in this study. The result of this research would provide the evidence-base practice in nursing as well as decline medical cost.