Neonatal septicemia is one of the major causes of morbidity and mortality worldwide during the neonatal period. It can be classified into two subtypes: early-onset sepsis (EOS) and late-onset sepsis (LOS) depending upon the time of onset. In the western developed countries, group B Streptococcal and Escherichia coli are leading pathogens for EOS, while the most frequent microorganism involved in LOS is coagulase negative Staphylococci, which are different from the domestic data. Clinical manifestations of neonatal septicemia are not specific, so that it is often misdiagnosed. This review describes the progress in diagnostic methods for neonatal septicemia, including blood culture, blood cell counts, cytokine profiles and umbilical cord blood examinations. It provides useful information for early diagnosis and treatment of neonatal septicemia.