Objective: The anastomosis of peripheral nerves is a demanding procedure that has potential complications due to foreign body reactions elicited by sutures. In this study, the sutureless in vivo anastomosis of rat tibial nerves was successfully performed, using for the first time a chitosan-based laser-activated adhesive. The nerve thermal damage caused by the laser irradiation was quantitatively assessed. Materials and Methods: A novel adhesive composed of chitosan, indocyanine green, acetic acid, and water, was fabricated in thin sheets. Its adhesive strength was tested in vitro by bonding strips (surface area ~20 mm², thickness ~20 μm) onto rat sciatic nerves and sheep intestine by laser activation with low fluence (~50 J/cm²), using a fiber-coupled diode laser (n = 13). The tensile strength of the adhesive/tissue bonds was measured after tissue repair. The chitosan adhesive was then used to perform sutureless anastomosis of tibial nerves in vivo (n = 6). Adhesive strips were also bonded in vivo onto intact rat sciatic nerves (n = 6) in order to quantitatively assess, by counting myelinated axons, the thermal damage induced by the laser. Results: The adhesive bonded well to tissue with a tensile strength of 12.5 ± 2.6 KPa (mean ± SD; n = 13). The in vivo anastomosed nerves were in continuity 3 d after surgery. Axon counting showed the number and morphology of myelinated axons were normal proximally (~96%) compared with intact nerves (100%). Axon demyelination was observed at the operation site (~49%) and distally (~27%), and was attributed to laser-induced thermal damage. Conclusions: Nerve anastomosis, performed by the laser-adhesive procedure, was successful 3 d postoperatively. Proximal myelinated axons were not significantly damaged by the low laser fluence.