The potential for using the enzyme pectinase as a pre-treatment to improve the properties of eucalyptus particle-based panels was investigated at different pre-treatment times, temperatures, and particle-to-enzyme ratios. As the pre-treatment time was increased from 10 min to 30 min, the free formaldehyde emission content was reduced (P < 0.0001). The pectinase enzyme pre-treatment reduced the pectin content and increased the permeability of treated wood, allowing more free formaldehyde to be released from the panels. The free formaldehyde emission content of all panels was lower than 3.0 mg per 100 g, due to the kind of urea-formaldehyde (UF) resin used. When the pre-treatment time was 30 min and the temperature was 45 °C, the mechanical properties, including modulus of elasticity (MOE), modulus of rupture (MOR), and internal bonding strength (IB), of the resulting panels were the best among all the selected treatment times and temperatures. As the ratio of particles to solution was reduced from 1:100 to 1:80 or 1:60, the mechanical properties of the particle board panels were improved (P < 0.0001). This was attributed to the pectinase enzyme pretreatment changing the surface of the particles, resulting in a better interface between UF resin and particles.