The false positive rate of the PPI test for the diagnosis of typical symptoms of gastroesophageal reflux disease (GERD) is extremely high. This study aims to investigate the effect of the pepsin test on GERD and laparoscopy-assisted anti-reflux surgery for GERD. A total of 30 GERD patients were enrolled into this study, and the pre-diagnosis of GERD was determined by symptom evaluation, impedance-pH examination, gastroscopy and pepsin test. All patients underwent surgery. Among the 30 GERD patients, 18 patients were male and 12 were female, and their average age was 58.2 + 12.6 years old. The patients were treated with laparoscopic fundoplication and hiatus hernia repair after preoperative assessment. A total of 28 patients were followed up, one patient developed recurrent symptoms, and one patient developed postoperative dysphagia and received non-operative treatment. Furthermore, the symptom scores were significantly lower at postoperative pepsin detection when compared to the scores before the operation (pepsin: preoperative: 148.8 ± 82.6, postoperative: 30.7 ± 24.6; t= 4.848, P= 0.000). Laparoscopic fundoplication and hiatus hernia repair may effectively control the symptoms of GERD. Furthermore, the detection of pepsin is non-invasive and easy to operate.