The partial substitution of chemical fertilizer with organic fertilizer is a crucial practice for enhancing crop production and quality, although its impact on natural rubber has rarely been explored. In this study, a two-year field experiment was conducted to investigate the impact of different nitrogen application rates and varying proportions of organic nitrogen substitution on dry rubber yield, nitrogen nutrition, and natural rubber properties. Regarding nitrogen application, the control treatment received no nitrogen amendment, while the low-nitrogen treatment was amended with 138 g·tree−1·year−1 of nitrogen. The medium-nitrogen treatment received 276 g·tree−1·year−1 of nitrogen, and the high-nitrogen treatment received 552 g·tree−1·year−1 of nitrogen. In addition, the low-organic-nitrogen substitution treatment and medium-organic-nitrogen substitution treatment were amended with 276 g·tree−1·year−1 of nitrogen each. The results demonstrated that the 50% organic nitrogen substitution treatment resulted in the highest dry rubber yield across all sampling periods, ranging from 46.43 to 94.65 g·tree−1. Additionally, this treatment exhibited superior soil total nitrogen (1067.69 mg·kg−1), available nitrogen (84.06 mg·kg−1), and nitrogen content in roots (1.08%), leaves (3.25%), fresh rubber latex (0.27%), and raw natural rubber (0.44%) compared with other treatments. In terms of the physical properties of natural rubber, the 50% organic nitrogen substitution treatment resulted in advantages in the weight-average molecular weight (1.57 × 106 g·mol−1), number-average molecular weight (0.36 × 106 g·mol−1), plasticity retention index (97.35%), Wallace plasticity (40.25), and Mooney viscosity (81.40). For mechanical properties, natural rubber from the substitution treatment exhibited higher tensile strength (19.84 MPa), greater elongation at break (834.75%), and increased tear strength (31.07 N·mm−1). Overall, the substitution of 50% chemical nitrogen fertilizer with organic nitrogen fertilizer improved nitrogen nutrition in rubber trees by introducing organic nitrogen input, resulting in remarkable enhancements in natural rubber properties. Therefore, the incorporation of organic fertilizer as a substitution for 50% of chemical fertilizer is demonstrated as an effective strategy for improving both the yield and properties of natural rubber.