Apolipoprotein A1 (Apo-A1) is a well-recognized biomarker in tissues, closely associated with cardiovascular diseases such as atherosclerosis, coronary artery disease, and heart failure. However, existing methods for Apo-A1 determination are limited by costly equipment and intricate operational procedures. Given the distinct advantages of electrochemical immunosensors, including affordability and high sensitivity, along with the unique attributes of nanobodies (Nbs), such as enhanced specificity and better tissue permeability, we developed an electrochemical immunosensor for Apo-A1 detection utilizing Nb technology. In our study, Ce-MOF@AuNPs nanocomposites were synthesized by using ultrasonic methods and applied to modify a glassy carbon electrode. The Nb6, screened from an Apo-A1 immunized phage library, was immobilized onto the nanocomposite material, establishing a robust binding interaction with Apo-A1. The recorded peak current values demonstrated a logarithmic increase corresponding to Apo-A1 concentrations ranging from 1 to 100,000 pg/mL, with a detection limit of 36 fg/mL. Additionally, the developed immunosensors demonstrated high selectivity, good stability, and reproducibility. Our methodology was also effectively utilized for serum sample analysis, showing good performance in clinical assessments. This electrochemical immunosensor represents a promising tool for Apo-A1 detection, with significant potential for advancing cardiovascular disease diagnostics.