Neutropenia is a common chemotherapy-derived complication in cancer patients, in whom the prevalence of sepsis ranges from 12.9 % to 17.4 %, with a lethality rate of 16 %. The aim of this study was to determine the usefulness of serum lactate as a biomarker of severe sepsis in children with cancer, fever and neutropenia. A phase II diagnostic test study was conducted. Lactate value was measured at admission. Neutropenia episodes were classified in three groups: I, with sepsis (patients with and without severe sepsis), II, without sepsis and III, neutropenic patients without fever (controls). Sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios were calculated. The gold standard was the clinical diagnosis of severe sepsis. A sample of 100 neutropenia episodes was collected: 16 were classified in group I, 73 in group II, and 11 in group III. Microbiological isolates were obtained in 11 samples, 4 from patients with severe sepsis and 6 from subjects without sepsis. A serum lactate level greater than or equal to 2 mmol/L had a sensitivity of 81 %, 83 % specificity, a positive predictive value of 48 % and a negative predictive value of 95 %; the positive and negative likelihood ratios were 4.88 and 0.23, respectively. According to the ROC curve, the area under the curve was 0.851 (95 % CI = 0.725-0.977). A serum lactate level greater than or equal to 2 mmol/L is consistent with severe sepsis in children with cancer, fever and neutropenia who are hemodynamically stable and without hypoperfusion.