Mattresses' pressure relief performance and comfort largely affect sleep quality. Mattress filling materials have been proven to affect the interface pressure distribution and comfort, but the effect of mattress structure is unclear. In this paper, the interface pressure distribution and subjective comfort of 10 subjects were assessed in the different bedding layer structures of mattresses, after mattress support performance was tested. The results show that the mattresses with bedding material hardness gradually increasing from the top layer to the bottom layer (BMH-ITTB) structure have a softer surface layer, a better support core layer, and higher fitness. This enables the mattress to achieve a better decompression effect. The low-pressure area (PAI≤0.67kPa) increased, while the high-pressure area (PAI≥2.67kPa and PAI≥4.00kPa), maximum pressure (P95), average pressure (P50), and pressure index (PI) decreased. This also enables the mattress to achieve higher subjective comfort scores.