Cells are normally cultured in 2D environment, which is usually inconsistent with the real microenvironment in vivo, and it is rarely reported that an effective cancer cell killing process occurs in a 3D network environment. Herein, a kind of new biomimetic composite hydrogel which can achieve 3D cell culture has been prepared and constructed by assembly of polyisocyanopeptide (PIC) with cationic oligo (p-phenylene vinylene) (OPV). The polymer chains of PIC can be bound and frizzled to form a 3D network when the temperature rises above the gelation temperature, followed by encapsulating the cells into biomimetic composite hydrogel. Cells grow and proliferate well in 3D composite hydrogels with excellent cell viability. When the cells undergo cancerization or microbial infection during the 3D culture, the addition of the luminol luminescence system can cause a strong bioluminescence resonance energy transfer (BRET) process to produce highly active reactive oxygen species (ROS) in 3D culture and kill the cancer cells and pathogenic microorganism effectively. Utilizing the BRET process in 3D composite biomimetic hydrogels provides an efficient antibacterial and anticancer approach in 3D culture to overcome the light-penetration limitation.