Background: The relationships between serum levels of homocysteine (Hcy), soluble stromelysin 2 (sST2), and tumor-associated cancer antigen 125 (CA-125) and heart failure requires further investigation. The aim of the present study was to evaluate the levels of Hcy, sST2 and CA-125 in patients with congestive heart failure and to correlate these with cardiac function, thereby providing a reference for the clinical diagnosis and treatment of heart failure. Methods: Seventy patients with chronic heart failure (CHF) diagnosed between August 2020 and July 2022 were classified into heart failure groups II (n = 25), III (n = 23) and IV (n = 22). Seventy individuals with normal physical examination results were selected as the healthy group. Serum Hcy, sST2 and CA-125 levels for all participants were evaluated and correlated with each other and with cardiac function classification. The diagnostic value of individual Hcy, sST2, CA-125 levels for CHF was evaluated, as well as a combination of these factors. Results: Hcy, sST2, and CA-125 levels were lower in the healthy group than in the heart failure group. Moreover, a progressive increase in Hcy, sST2, and CA-125 levels were observed in heart failure groups II, III, and IV. Individual Hcy, sST2 and CA-125 levels, as well as a combination of these factors, were significantly correlated with cardiac function classification (p < 0.05). Hcy, sST2 and CA-125 levels each showed diagnostic value for CHF, with the three combined having the best diagnostic value. Conclusions: Abnormally high levels of Hcy, sST2 and CA-125 occur in CHF patients and are positively correlated with cardiac function classification. Individual levels of these factors, and particularly a combination of the three, show good sensitivity and specificity for CHF diagnosis that could be widely used in clinical practice.