A simple and feasible electrochemical immunosensing protocol with glucometer readout was designed for the detection of low-abundance disease-related biomarker (alpha-fetoprotein; AFP) on the basis of backfilling rolling cycle amplification (RCA) with invertase-DNA2 conjugates on the detection antibody. The assay consisted of the immunoreaction, RCA reaction, DNA2-invertase hybridization and glucose measurement. Initially, a sandwiched immunoreaction was carried out between anti-AFP capture antibody-coated microplate between nanogold-labeled pAb2 detection antibody conjugated with DNA1 primer (DNA1-AuNP-pAb2) in the presence of target ATP. Thereafter, the carried primers triggered the RCA reaction in the presence of circular DNA template, polymerase and dNTP, to produce numerous repeated oligonucleotide sequences for hybridization with many invertase-DNA2 conjugates. The carried invertase molecules accompanying the hybridization reaction hydrolyzed sucrose into glucose, thereby resulting in the amplification of the detectable signal on a handheld personal glucometer (PGM). Under optimum conditions, the developed immunoassay exhibited high sensitivity for the quantitative screening of AFP within a dynamic range of 0.1–100 ng mL−1 at a low detection limit of 0.087 ng mL−1. Other biomarkers and proteins did not interfere the signals of this system. In addition, this method was utilized to determine human serum samples containing target AFP, and received well-matched results with the referenced enzyme-linked immunosorbent assay (ELISA) method.