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The diagnosis of spontaneous bacterial peritonitis (SBP) depends primarily on a polymorphonuclear leukocyte cell count more than 250/mm3. This method is invasive, and not diagnostic in all variants of SBP; we aimed to assess serum homocysteine as a precise indicative marker for the diagnosis of all variants of SBP.A total 323 consecutive ascitic patients were registered in this prospective work. Serum and ascitic fluid of homocysteine were evaluated utilizing an enzyme-linked immunosorbent assay.Participants were classified into a non-SBP group, including 262 participants and 61 patients with SBP. Serum and ascitic homocysteine were considerably elevated in the SBP group than in the non-SBP group (17.94±7.57 vs. 11.75±5.68 μmol/l; P<0.001 and 14.70±5.45 vs. 9.75±4.55 μmol/l; P<0.001). At a cutoff value of 17.79 μmol/l, serum homocysteine had 89.3% specificity and 95.1% sensitivity for distinguishing SBP (area under the curve: 0.932) and, at a cutoff value of 16.1 μmol/l, ascitic homocysteine had 84.4% specificity and 92.7% sensitivity for distinguishing SBP (area under the curve: 0.901). Both were positively correlated with the polymorphonuclear count, C-reactive protein, Child–Pugh score, and Model For End-Stage Liver Disease score as well as negatively correlated with the protein content in the ascitic fluid and estimated glomerular filtration rate. After SBP therapy, there was a marked reduction in serum and ascitic homocysteine levels.This study demonstrates that serum and ascitic homocysteine are considerably higher in SBP participants versus non-SBP patients. Serum homocysteine may provide a reliable and noninvasive diagnostic marker for all variants of SBP.