Purpose [F-18]-D3FSP is a new beta-amyloid (A beta) PET imaging tracer designed to decrease nonspecific signals in the brain by reducing the formation of the N-demethylated product. However, its optimal reference region for calculating the standardized uptake value ratio (SUVR) and its relation to the well-established biomarkers of Alzheimer's disease (AD) are still unclear. Methods We recruited 203 participants from the Greater Bay Area Healthy Aging Brain Study (GHABS) to undergo [F-18]-D3FSP A beta PET imaging. We analyzed plasma A beta 42/A beta 40, p-Tau181, glial fibrillary acidic protein (GFAP), and neurofilament light (NfL) using the Simoa platform. We compared the standardized uptake value (SUV) of five reference regions (cerebellum, cerebellum cortex, brainstem/PONs, white matter, composite of the four regions above) and AD typical cortical region (COMPOSITE) SUVR among different clinical groups. The association of D3FSP SUVR with plasma biomarkers, imaging biomarkers, and cognition was also investigated. Results Brainstem/PONs SUV showed the lowest fluctuation across diagnostic groups, and COMPOSITE D3FSP SUVR had an enormous effect distinguishing cognitively impaired (CI) individuals from cognitively unimpaired (CU) individuals. COMPOSITE SUVR (Referred to brainstem/PONs) was positively correlated with p-Tau181 (p < 0.001), GFAP (p < 0.001), NfL (p = 0.014) in plasma and temporal-metaROI tau deposition (p < 0.001), and negatively related to plasma A beta 42/A beta 40 (p < 0.001), temporal-metaROI cortical thickness (p < 0.01), residual hippocampal volume (p < 0.001) and cognition (p < 0.001). The voxel-wise analysis replicated these findings. Conclusion This study suggests brainstem/PONs as an optimal reference region for calculating D3FSP SUVR to quantify cortical A beta plaques in the brain. [F-18]-D3FSP could distinguish CI from CU and strongly correlates with well-established plasma biomarkers, tau PET, neurodegeneration, and cognitive decline. However, future head-to-head comparisons of [F-18]-D3FSP PET images with other validated A beta PET tracers or postmortem results are crucial.