Abstract: This study aims to investigate the heterodimeric probe ¹⁸FAlF-labeled targeting both fibroblast activation protein (FAP) and integrin receptor α_Ⅴβ₃: 1,4,7-triazacyclononane-1,4,7-triacetic acid-FAP inhibitor-arginine-glycine-aspartic acid (NOTA-FAPI-RGD) for its potential application in small-animal PET imaging of FAP-positive tumor-bearing nude mouse models and its preliminary clinical application. ¹⁸FAlF-NOTA-FAPI-RGD was synthesized using an optimized ¹⁸FAlF-labeling method developed in previous studies. Cellular uptake and blocking experiments were performed in HT1080-FAP tumor cells to evaluate specific uptake in vitro. PET imaging was conducted in HT1080-FAP tumor-bearing mice to observe the distribution of ¹⁸FAlF-NOTA-FAPI-RGD in the HT1080-FAP tumor model. Competitive PET imaging was performed by co-injection of NOTA-FAPI-02, NOTA-RGD, and NOTA-FAPI-RGD inhibitors, respectively, alongside imaging using ¹⁸FAlF-NOTA-FAPI-02 and ¹⁸FAlF-NOTA-RGD, to assess dual-target specificity in vivo. In addition, preliminary clinical PET imaging was also performed in breast cancer patients to evaluate its diagnostic performance. The dual-target radiotracer ¹⁸FAlF-NOTA-FAPI-RGD is successfully synthesized and evaluated in this study. It exhibites significant uptake in HT1080-FAP tumor cells; Cell uptake can reach up to 44.66%±0.26%. After being blocked with NOTA-FAPI-02, NOTA-RGD, and NOTA-FAPI-RGD inhibitors, the cell uptake decreases to 0.46%±0.04%, 37.61%±1.21%, and 0.16%±0.02%, with P<0.01, which confirms the dual-target specificity of ¹⁸FAlF-NOTA-FAPI-RGD. In HT1080-FAP tumor-bearing mice, PET imaging demonstrates that ¹⁸FAlF-NOTA-FAPI-RGD displays excellent pharmacokinetics, with high tumor uptake and prolonged retention time: Tumor uptake can reach up to (9.67±1.23)%ID/g at 0.5 h, and 6 h post-injection, tumor uptake still retains (8.10±1.35)%ID/g. Tumor uptake is effectively inhibited by NOTA-FAPI-02, NOTA-RGD, and NOTA-FAPI-RGD inhibitors, which demonstrates the dual-targeting specificity for both FAP and integrin receptor α_Ⅴβ₃. In contrast, the control radiotracers ¹⁸FAlF-NOTA-FAPI-02 and ¹⁸FAlF-NOTA-RGD show lower HT1080-FAP tumor uptake and retention, which indicates the superiority of ¹⁸FAlF-NOTA-FAPI-RGD. Furthermore, clinical PET imaging in breast cancer patients demonstrates a greater lesion uptake value for ¹⁸FAlF-NOTA-FAPI-RGD (SUV_max=12.41) compared to ¹⁸FFDG (SUV_max=7.45). ¹⁸FAlF-NOTA-FAPI-RGD demonstrates dual-targeting specificity for both FAP and integrin receptor α_Ⅴβ₃, as well as high tumor uptake and prolonged tumor retention in both preclinical and clinical studies. Compared to single-targeting tracers, it shows superior imaging quality and lesion detection. These results highlight the potential of ¹⁸FAlF-NOTA-FAPI-RGD as a radiotracer for diagnosing tumors with high FAP and/or integrin α_Ⅴβ₃ expression, which offers promising prospects for clinical application.