In recent years, the issues of declining male fertility and increasing incidence of testis-related diseases have become increasingly prominent, with major contributing factors including environmental toxins, genetic factors, and unhealthy lifestyles. Meanwhile, in the safety evaluation of drugs and environmental compounds, male reproductive toxicity assessment has emerged as a criticalcomponent. Traditional animal experiments are limited by long cycles, ethical controversies, and interspecies differences, while two-dimensional (2D) cell culture struggles to simulate the complex in vivo microenvironment of the testis. With the promotion of the 3R principles (Reduction, Replacement, Refinement), the development of in vitro models with high physiological relevance has become an inevitable trend in reproductive toxicology research. Three-dimensional (3D) cell culture can mimic the interactions between the extracellular matrix and cells, providing a suitable physiological niche for testicular cells. This article systematically reviews the research progress of 3D culture technology in the development of testicular models. Firstly, it elaborates on the composition of the testicular germ cell niche and its significance in toxicity assessment. Secondly, from the perspectives of technical principles and applications, it analyzes the characteristics and performance of five culture systems: decellularized testicular scaffolds, hydrogel scaffolds, nanofiber scaffolds, organoids, and 3D bioprinting. Finally, identifies the discrepancies and limitations of existing models, and proposes that future research should focus on areas such as model standardization, optimization of multicellular co-culture systems, and integration of high-throughput screening technologies. These efforts aim to facilitate the widespreadapplication of 3D testicular models in reproductive toxicity research, environmental safety assessment, and pharmaceutical research and development.