Irregular hepatectomy plays a prominent role in the treatment of small hepatocellular carcinoma (HCC) patients with severe cirrhosis and localized liver metastasis. In clinical practices, intraoperative tumor boundaries delineation facilitates to accomplish tumor resection with negative margin, remarkably decreasing the recurrence rates. Currently, ultrasound (US) and ICG fluorescence-guided surgery has been used for intraoperative navigation in irregular hepatectomy, but insufficient specificity results in a limited prevalence. Inspired by the high resolution of photoacoustic (PA) imaging and established clinical efficacy of 18F-Alfatide that is specific for integrin αvβ3-overexpressed tumors, we herein developed a fluorescent analogue IR820-E[c(RGDfK)]2, and a proof-of-concept intraoperative multispectral PA imaging navigation for precise irregular hepatectomy using hand-held PA/US imaging system. An integrin αvβ3-targeted fluorescent contrast agent IR820-E[c(RGDfK)]2 was designed, synthesized, and characterized. In vitro studies were performed to determine optical and PA properties, affinity and specificity and biocompatibility. Multispectral PA imaging, the optimal imaging time point and contrast, multispectral PA imaging-guided irregular hepatectomy, pharmacokinetics, and safety profile were evaluated in subcutaneous and orthotopic HCC tumor models. Ex vivo macroscopic three-dimensions (3D) PA imaging with IR820-E[c(RGDfK)]2 staining was also performed in surgical biospecimens from patients with HCC. IR820- E[c(RGDfK)]2 has a simple synthetic method at gram scale, high affinity, and specificity for integrin αvβ3, excellent pharmacokinetic and safety profile can effectively differentiate tumor from normal liver tissues in animal models and surgical biospecimens from HCC patients. Preoperative tumor localization, intraoperative tumor boundaries delineation, and tumor excision, and postoperative negative margin assessment were successfully achieved during irregular hepatectomy. This initial attempt allows one to preoperatively detect tumor lesions, intraoperatively delineate tumor boundaries and guide tumor resection, and postoperatively evaluate tumor margin status during irregular hepatectomy. IR820-E[c(RGDfK)]2 has the potential to be an investigational new drug for clinical use in multispectral photoacoustic imaging-guided irregular hepatectomy.