This study investigates the effects of various production parameters on the physical and mechanical properties of one-layer particleboards bonded with canola protein-based adhesives. Two protein-based adhesive formulations, CPI-B-0 with sodium bisulfate and CPI-N-60 with sodium nitrite crosslinkers, were examined under different conditions: binder content, press temperature, and press time factor. Results indicate that the CPI-N-60 outperformed the CPI-B-0 in terms of internal bonding strength (IB), modulus of rupture (MOR), and modulus of elasticity (MOE) due to the stronger covalent bonds formed with primary amines present in the protein adhesive. Increasing binder content led to significant improvements in mechanical properties, with the internal bonding and the MOR increasing by 21% and 9% when using 9% binder content over 7% respectively. The press temperature, as well as the press time were found to yield the highest influence on mechanical properties, with higher values resulting in better performance. Increasing the press temperature from 170 °C to 190 °C led to 33% increment in the internal bonding and 20% in the MOR, while 67% and 28% increment was obtained with 210 °C press temperature, respectively. Increasing the press time also led to an increase in the mechanical properties of the particleboards, by almost the same proportions as the effect of press temperature. The interaction effects between production parameters highlighted the importance of optimized conditions for achieving the desired properties. Indeed, under certain press conditions, the CPI-N-60 outperformed the conventional UF K345, achieving an IB value of 0.8 N/mm2 over 0.65 N/mm2. Overall, this study contributes to a better understanding of canola protein-based bio adhesive, with implications for the optimization of the production parameters for better boards’ properties.