Using serum biomarkers that reflect fruit and vegetable (FV) intake offers a significant advantage over traditional dietary assessments by providing a more objective, accurate measure, meaningfully minimizing recall bias and misreporting common in self-reported dietary data. This study investigated the relationship between these serum biomarkers and mortality risk using data from 19,168 adults aged 30 and older who participated in the National Health and Nutrition Examination Survey (NHANES) from 1988 to 2006. Mortality follow-up was determined by linkage to the National Death Index through December 31, 2019, and diet by 24-h recalls. Cox proportional hazards models were employed to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for mortality outcomes by tertiles of serum biomarkers of FV intake. Higher serum concentrations of total carotenoids were associated with a reduced risk of all-cause (Tertile 3 vs. Tertile 1 HR=0.69, 95% CI=0.61-0.78) and cancer mortality (HR=0.53, 95% CI=0.39-0.71). Greater serum concentrations of individual carotenoids, such as α-carotene, β-carotene, β-cryptoxanthin, lycopene, lutein and zeaxanthin, were also linked to lower risks of all-cause and cancer mortality. Higher serum potassium concentrations showed a trend toward an association with a greater risk of all-cause mortality. No significant associations were found between serum vitamin C concentrations and mortality outcomes in the overall analysis; however, in sex-stratified analyses, higher vitamin C concentrations were associated with reduced risk of all-cause and cancer mortality in women. These findings suggest that specific serum biomarkers of FV intake, particularly carotenoids and vitamin C, may serve as indicators of reduced mortality risk.