An eco-friendly medium-density fiberboard (MDF) was prepared using vanillin (V) crosslinked chitosan (CS) adhesive through a hot-pressing process. The cross-linking mechanism and the effect of different proportions of added chitosan/vanillin on the mechanical properties and dimensional stability of MDF were investigated. The results showed that vanillin and chitosan are crosslinked to form a three-dimensional network structure due to the Schiff base reaction between the aldehyde group of vanillin and the amino group of chitosan. At the same time, when the mass ratio between vanillin/chitosan was 2:1, MDF obtained the best mechanical properties, the maximum modulus of rupture (MOR) of 20.64 MPa, the mean modulus of elasticity (MOE) of 3005 MPa, the mean internal bonding (IB) of 0.86 MPa, and the mean thickness swelling (TS) of 14.7%. Therefore, the MDF bonded with V-crosslinked CS can be a promising candidate for environmentally-friendly wood-based panels.