Background: Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in modern human life. Serum protease and protease inhibitor concentrations can be potentially considered the important risk factors for NAFLD, together with the main risk factors of this disorder, such as diabetes, obesity, and dyslipidemia. Objectives: The present study aimed to investigate the plasma level of a neutral serine protease family called neutrophil elastase (NE), as well as its inhibitor, alpha1-antitrypsin (A1AT), in NAFLD patients compared to normal subjects. Additionally, the most common deficient variants of A1AT (S and Z) were determined in both patient and healthy groups. Methods: The study included 54 consecutive unrelated NAFLD patients and 120 matched healthy subjects as controls over two years. Plasma concentrations of A1AT and neutrophil elastase activity were determined for all patients and controls using the enzyme immunoassay and alpha1-proteinase inhibitor-immunoglobulin A complex, respectively. The A1AT variants of PiZ (rs28929474) and PiS (rs17580) were analyzed by the polymerase chain reaction. Results: The NAFLD patient group had more plasma elastase activity than the healthy control group (1.7 (0.5) vs. 1.3 (0.5) (U/mL), P < 0.01). Furthermore, the plasma A1AT level was significantly lower in NAFLD patients than in controls (216.1 (171.3) vs. 244.6 (152.9) (mg/dL), P = 0.01). The heterozygous carriages of PiS and PiZ variants were not statistically different between NAFLD patients and controls. Conclusions: Higher elastase activity and lower A1AT concentration in plasma can be considered the potential prognostic and diagnostic biomarkers in patients with NAFLD.