Background Current ways to diagnose citrate accumulation (CA) in patients receiving regional citrate anticoagulation (RCA) continuous renal replacement therapy (CRRT) are confounded by various clinical factors. Serum citrate measurement emerges as a more direct way to diagnose CA, but its clinical utility and optimal cut-off values remain undefined. This study examined serum citrate kinetics and its diagnostic performance for CA in patients receiving RCA CRRT. Methods A multi-center prospective study carried out in 2 tertiary referral center ICUs in Hong Kong with serum citrate levels measured at baseline, 2-, 6-, 12-, 24-, 36-, 48- and 72-hours after initiation of RCA CRRT and their relationships with development of CA examined. Results Amongst the 133 patients analyzed, 18 patients (13.5%) developed CA. The serum citrate levels at baseline, 2-, 6- and 12-hours after initiation of RCA CRRT in patients who had CA were significantly higher than the non-CA group (P < 0.001, for all). The CA group also had higher serum citrate levels than the non-CA group [median (IQR): 0.93(0.81–1.16) mmol/L vs. 0.37(0.26–0.57) mmol/L, P < 0.001]. Using a cut-off of 0.85 mmol/L, serum citrate level had a sensitivity (SN) of 0.77 and a specificity (SP) 0.96 for the diagnosis of CA (AUROC 0.90, P < 0.001). The 2-hr and 6-hr serum citrate levels had good discriminatory abilities for predicting subsequent development of CA (AUROC 0.86 and 0.83 for 2-hr and 6-hr citrate levels using cut-off values of 0.34 and 0.63 mmol/L respectively; P < 0.001). Conclusion Serum citrate levels were significantly higher in patients with CA compared with patients without CA. Serum citrate levels showed good performance in diagnosing and predicting the development of CA.