Objective To study the serum level of cystatin C (Cys C) and plasma level of N-terminal pro-brain natriuretic peptide (NT-pro BNP) in the heart failure patients with preserved ejection fraction (HFpEF) and those with reduced ejection fraction (HFrEF), and investigate their relationships and clinical significances. Methods Seventy-five patients (≥60 years old) with established diagnosis of heart failure (at class Ⅱ, Ⅲ and Ⅳ respectively according to NYHA classification) admitted to Cadre’s Ward and Department of Cardiology in our hospital from January 2015 to June 2016 were recruited in this study. They were 55 cases of HFpEF and 20 cases of HFrEF. Another 50 patients (≥60 years old) without the history of cardiovascular disease served as controls. The clinical data were collected and compared in the 3 groups. Serum Cys C level, plasma NT-Pro BNP level and left ventricular ejection fraction (LVEF) were measured and their correlations were analyzed. The values of Cys C and NT-pro BNP in the diagnoses of 2 types of heart failure were investigated with receiver operating characteristic (ROC) curve. Results The patients of the HFpEF group were older than those from HFrEF group and control group (P<0.05). There were significant differences in the levels of Cys C and NT-Pro BNP among the 3 groups (P<0.05). The Cys C level of the HFpEF group was positively correlated with the levels of NT-Pro BNP, creatinine, blood urea nitrogen and uric acid (P<0.05), and the level was still correlated with that of NT-pro BNP after adjustment for the interference factors (r=0.460, P=0.001). The level of NT-pro BNP was negatively correlated with LVEF in the HFpEF group and HFrEF group (r=-0.277, P=0.041; r=-0.512, P=0.021). The ROC curve analyses showed that the cut-off level of NT-pro BNP was 355 ng/L for HFpEF diagnosis and 1835 ng/L for HFrEF diagnosis with the highest Youden’s indices and the sensitivity of 98.2% and 95.0% respectively, while the specificity of 60.0% and 78.1% separately. The cut-off level of Cys C was 1.765 mg/L for HFrEF, with the largest area under ROC curve and the sensitivity of 85.0% while the specificity of 75.2%. Conclusion The serum level of Cys C and plasma level of NT-pro BNP are different between the elderly heart failure patients with HFpEF and those with HFrEF. NT-pro BNP is a sensitive biomarker in the diagnosis of heart failure, and Cys C is more significant in the diagnosis of HFrEF than that of HFpEF.