The work aims to study the effect of tree species and heartwood-sapwood content on the physical and mechanical properties of ultra-thin high-density fiberboard. Ultra-thin high-density fiberboard was prepared with fibers from different tree species (poplar, eucalyptus and pine) and heartwood-sapwood fibers from large-diameter poplar as raw materials and phenol formaldehyde (PF) resin as the adhesive. The effects of different tree species and heartwood-sapwood contents on the physical and mechanical properties of the fiberboard were investigated in depth. Based on the experimental results, the mathematical model of the relationship between heartwood content, sapwood content and the physical and mechanical properties of ultra-thin high-density fiberboard was established. Among different tree species, the physical and mechanical properties of poplar fiberboard were the best, with a bending strength of 54.61 MPa, an elastic modulus of 4.967 GPa, a water absorption thickness swelling rate of 27.87%, and a surface roughness of 2.361 μm. Within the range of this experiment, when the heartwood content was 30% and the sapwood content was 50%, the physical and mechanical properties of the ultra-thin high-density fiberboard were the best, with a bending strength of 57.69 MPa, an elastic modulus of 5.812 GPa, a water absorption thickness swelling rate of 27.43%, and a surface roughness of 2.719 μm. The physical and mechanical properties of the above fiberboard all exceeded the performance standards stipulated in "Ultra-thin High-density Fiberboard" (T/CNFPIA 3007-2019). The tree species and heartwood-sapwood content of the fiberboard can be adjusted according to actual needs to prepare ultra-thin high-density fiberboard products that meet the production process and usage requirements.