Neonatal sepsis is a severe systemic infection for which timely and accurate diagnosis is crucial to reduce mortality and improve patient outcomes. While traditional inflammatory markers such as C-reactive protein and white blood cell count are widely used in clinical diagnosis, their limitations in specificity and sensitivity can lead to diagnostic delays or errors. With advancements in molecular biology and immunology, a range of novel molecular markers—including cytokines, microRNAs, and metabolomic biomarkers—have been identified, offering new possibilities for the early diagnosis of neonatal sepsis. This review summarizes the current research on both traditional inflammatory markers and novel molecular markers for the diagnosis of neonatal sepsis, analyzes their advantages and limitations, and explores their clinical application potential and future directions. The aim is to provide a theoretical basis and practical guidance for optimizing clinical diagnostic strategies and enabling personalized treatment.