Infection is the main problem among the patients receiving cancer therapy. The mortality rate can be reduced by the appropriate treatment in the right time. Although blood culture is the gold standard for the diagnoses of sepsis, many factors influence the results of blood culture in children. For this reason, real time polymerase chain reaction (Rt-PCR) has gained importance for the diagnoses of microbiological agents as it is faster than the conventional methods. In this study, we aimed to compare the efficacy of SeptiFast (SF) test with blood culture among children with neutropenic fever. Between January 2013 and December 2014, 62 children (34 boys, 38 girls) mean age 7.56 ± 4.8 (0-18) years with cancer were included in this study during their 94 febrile attacks of neutropenia (NA). Blood samples for blood culture and Septifast test were taken before the initiation of antibiotic therapy. Blood cultures were routinely collected in aerobic and anaerobic media and incubated using the BACTEC 9120 system (Becton-Dickinson Diagnostic Systems, USA). Identification and antimicrobial susceptibility testing of the isolates were performed using the Vitek2® system (bioMérieux, France) according to the recommendations of the Clinical and Laboratory Standards Institute. The LightCycler SF test was used according to the manufacturer instructions. Of 94 attacks 34 (36.1%) were positive for blood culture and 33 (35.1%) for SF test. The positivity ratio is found as 29.7% (28/94) by blood culture when the analysis of five coagulase negative staphylococci were excluded due to contamination. Positivity was detected in 25 (26.6%) of the 94 NA both with blood culture and SF test.The difference between these two tests was statistically significant (p< 0.05). There was discordance with a rate of 28.7% between these two methods. Polymicrobial infections were detected only with SF test. The detection of fungal infection rate was higher with SF test than blood culture. When SF test was compared with blood culture the results were as follows; sensitivity 91%, specificity 98.3%, positive predictive value 97%, negative predictive value 96.7%, diagnostic performance was 96.2%, respectively. As a result, PCR based tests can be used in children with NA attacks even though blood culture is still needed to perform the antibiotic sensitivity tests. SF test seems to be a sensitive test for the early diagnosis of the pathogens and the initiation of the appropriate therapy according to the etiological agent.