Bio-based polyol (ester, ether) are used for the preparation of polyurethane (PU) adhesives, which were synthesized from tobacco stem and nontraditional oil by transesterification reaction. These prepared polyols were reacted with isocyanates adduct to form a PU adhesive, where different NCO/OH ratios were used to give various compositions. The polyols and PUs were characterized by FTIR spectroscopy and further TGA was used for the analysis of thermal properties. The tobacco stem based PU adhesives had comparable or better adhesive properties in terms of lap shear strength than the commercial adhesives. The effect of the NCO/ OH ratio on adhesive characteristics on wood bonding was also evaluated by lap shear tests. The change in lap shear strength before and after exposure to cold water, hot water, acid, and alkali were evaluated. The resultant formulated PU adhesive from agricultural waste was comparable to the commercially available adhesive.