Chimeric antigen receptor T-cell (CAR-T) therapy has demonstrated remarkable clinical efficacy in certain hematologic malignancies. However, its broader clinical application is constrained by the prolonged duration of conventional ex vivo manufacturing. The extended production cycle not only delays treatment and increases costs but may also exacerbate terminal differentiation and functional exhaustion of T cells, thereby reducing their in vivo persistence. To address these bottlenecks, shortening the ex vivo culture duration to preserve the early differentiation phenotype and antitumor activity of T cells has emerged as an important strategy for optimizing CAR-T cell therapy. In this study, a rapid CAR-T cell production process requiring only 24 h was established. Through systematical optimization of T-cell activation conditions and the viral transduction time window, efficient CAR gene delivery and cell preparation were achieved within 24 h. The rapidly manufactured CAR-T cells exhibited an early differentiation phenotype and enhanced proliferative potential. In vitro functional assays demonstrated potent tumor-killing activity of the CAR-T cells. In a xenograft mouse model, the cells showed antitumor efficacy and in vivo persistence comparable to those produced with the conventional manufacturing process. These results indicate that the 24 h rapid production strategy is feasible and provides a promising technical approach to shorten patient waiting time, reduce production costs, and generate functionally improved CAR-T cell products for clinical translation. 嵌合抗原受体(chimeric antigen receptor, CAR) T细胞疗法已成为部分血液恶性肿瘤的有效治疗手段，但其应用仍受到传统体外制备周期的限制。该周期不仅导致治疗延迟和成本增加，还可能导致T细胞终末分化加剧、功能衰竭和体内持久性减弱。为克服上述瓶颈，缩短体外培养时间以维持T细胞的早期状态和抗肿瘤活性，成为优化CAR-T疗法的重要研究方向。本研究开发了一种仅需24 h的CAR-T细胞快速制备工艺。该工艺通过对T细胞激活条件及病毒转导窗口进行系统性调整分析并优化筛选，实现了24 h内的高效CAR基因递送与细胞制备，使所得的CAR-T细胞产品呈现早期分化表型，具有更强的增殖潜能。体外功能实验表明，快速制备的CAR-T细胞具备强大的肿瘤杀伤能力。在异种移植瘤小鼠模型中具备传统制备工艺相当的抗肿瘤活性与持久性。本研究证实了24 h快速制备工艺的可行性，为缩短患者等待时间、降低生产成本、获得功能更优的CAR-T产品提供了新的解决方案。.