Objective To explore the feasibility of constructing tissue engineering cartilage with acellular cartilage matrix (ACM) biomimetic scaffolds in vitro. Methods The cartilage tissue was decellularized after completely pulverization, and then combined with a certain proportion of gelatin (GT) to prepare three-dimensional porous scaffolds. The pore size, porosity, biomechanics and degradation rate of porous scaffolds with different ACM/GT ratios were analyzed, and the most suitable proportion for cartilage regeneration in vitro was selected. Porcine articular cartilage cells were cultured, and then inoculated into ACM scaffolds. Gross observation and histological staining were carried out after culturing for 8 weeks in vitro. Results With the increase of ACM content, the pore size and porosity of ACM scaffolds increased while the biomechanical strength decreased. In terms of pore size and porosity, the ACM:GT=5:5 one was suitable for cartilage reconstruction. Gross observation and histology showed that ACM scaffolds could reconstruct homogeneous and typical cartilage tissue in vitro. Conclusion ACM can be used to fabricate three-dimensional porous scaffolds that are suitable for cartilage regeneration and to construct tissue engineering cartilage in vitro.