Several metabolites in human serum have been identified as potential cancer biomarkers for early detection. This study focuses on the LC–MS/MS method development and validation of D-mannose in human serum. Surrogate blank serum, coupled with stable isotope D-mannose-13C6, as internal standard, was used for generating standard curves ranging from 1 to 50 μg/mL. Separation was achieved by an Agilent 1200 series HPLC equipped with a SUPELCOGELTM Pb, 6% Crosslinked column with HPLC water as a mobile phase at flow rate of 0.5 mL/min at 80 °C. Mass detection was performed under negative ionization electrospray. Inter- and intra-day accuracy and precision were <2%. The extraction recovery and matrix effect were 104.1%–105.5% and 97.0%–100.0%, respectively. This method was successfully applied for the quantification of D-mannose in the serum samples of 320 esophageal cancer patients and 323 healthy volunteers. We report a simple, specific and reproducible LC–MS/MS method for the quantification of D-mannose in human serum as a potential cancer biomarker.