Purpose Laparoscopic liver resection is increasingly used to treat patients with hepatic tumor. However, during laparoscopic resection, information obtained by palpation using laparoscopic forceps or from intraoperative ultrasonography is extremely limited, which may pose a risk for positive surgical margins. This study aimed to investigate the feasibility and clinical application of near-infrared (NIR) fluorescence imaging to guide laparoscopic liver resection of a liver tumor and secure the surgical margins. Methods A preliminary study in 25 patients was conducted. NIR imaging was used intraoperatively during laparoscopic liver resection. The liver tumors were preoperatively labeled by intravenously injecting the patients with indocyanine green dye (0.5 mg/kg), an NIR fluorescence agent. During the surgical procedure, the PINPOINT Endoscopic Fluorescence Imaging System was used to assess the surgical margin by using real-time endoscopic high-definition visible and NIR fluorescence imaging. Results All tumors were identified and resected laparoscopically by using the PINPOINT system, and all resections successfully secured the surgical margin. The pathological findings of all tumors indicated negative margins, defined as R0. Conclusions This technique showed the potential to improve the intraoperative identification and demarcation of tumors. Its use could potentially reduce the number of positive resection margins.