The blood proteome contains biomarkers of ageing and age-associated diseases, but such markers are rarely validated longitudinally. Here we map the longitudinal proteome in 7,565 serum samples from a cohort of 3,796 middle-aged and elderly adults across three time points over a 9-year follow-up period. We pinpoint 86 ageing-related proteins that exhibit signatures associated with 32 clinical traits and the incidence of 14 major ageing-related chronic diseases. Leveraging a machine-learning model, we pick 22 of these proteins to generate a proteomic healthy ageing score (PHAS), capable of predicting the incidence of cardiometabolic diseases. We further identify the gut microbiota as a modifiable factor influencing the PHAS. Our data constitute a valuable resource and offer useful insights into the roles of serum proteins in ageing and age-associated cardiometabolic diseases, providing potential targets for intervention with therapeutics to promote healthy ageing.