The lack of a specific biomarker that can be used in the diagnosis of acute cholecystitis, a common cause of admission to the emergency department, delays physician efforts to diagnose and treat these patients. Therefore, the aim of this study was to measure plasma YKL-40 levels and investigate their diagnostic value in patients with acute cholecystitis (AC). This study was carried out between February 2020 and September 2020 in the adult emergency department of a tertiary university hospital. Permission was obtained from the Ethics Committee of Scientific Research on 03/02/2020 with Decision No. 03/16. The study included 80 patients who were diagnosed with acute cholecystitis and 80 healthy volunteers without known chronic diseases. The median YKL-40 protein level was 798.66 pq/mL in the patient group and 392.45 pq/mL in the control group. A statistically significant difference in YKL-40 protein levels was found between the two groups. YKL-40 protein levels were significantly higher in patients diagnosed with acute cholecystitis than in healthy individuals (p < 0.001). A positive correlation was found between YKL-40 protein levels and ALT, AST, LDH, and GGT levels (r = 0.272, p = 0.015; r = 0.397, p < 0.001; r = 0.386, p < 0.001; and r = 0.264, p = 0.018; respectively). When evaluated together with physical examination, radiological imaging and other laboratory parameters, we think that plasma YKL-40 levels can be used effectively in the diagnosis of acute cholecystitis.