Resorcinol-formaldehyde (RF) resin is a kind of excellent exterior-grade wood structural adhesive, which can be conveniently cold-set for various applications. In order to decrease the production cost, pyrolysis bio-oil from renewable bioresources was used to replace resorcinol to synthesize the bio-oil-resorcinol-aldehyde (BRF) resin. The effect of replacing resorcinol with bio-oil on the properties, bonding performance, and characterization of resorcinol-aldehyde resin was comparatively investigated. A higher solid content and viscosity, albeit a lower shear strength, was found when the replacement ratio of bio-oil increased. The bonding performance of BRF with 10 and 20 wt % bio-oil was close to that of the pure RF resin. However, the trends of being less cross-linked, more easily decomposed, but more porous were found when the substitution ratio of bio-oil was higher than 20 wt %. Interestingly, it was found that the wood failure values of the BRF resins with bio-oil of no more than 20 wt % were slightly higher than that of the pure RF resin. On the whole, BRF resins with 20 wt % bio-oil is recommended as a wood structural adhesive, comprehensively considering the bio-oil substitution ratio and resin properties. The results obtained here showed that pyrolysis bio-oil is a promising green raw material for the production of RF resin with lower cost.