To detect high-sensitivity cardiac troponin I (hs-cTnI; <0.01 ng/mL) at points of care, we developed a rapid immunosensor by using horseradish peroxidase polymerized in 20 molecules on average (Poly-HRP) as a tracer conjugated with streptavidin (SA-Poly-HRP). As shown in the conventional system, enhanced sensitivity could be achieved by using a sequential binding scheme for the complex formation to contain the huge molecular tracer. We used a 2-dimensional chromatographic technology to carry out the sequential bindings in cross-flow directions. After the complex formation of antigen-antibody with analyte in a vertical direction, SA-Poly-HRP was horizontally supplied across the membrane strip for additional binding via a biotin-SA linkage. The HRP substrate was subsequently supplied along the same direction to produce a chemiluminometric signal, which was measured by a cooled charge-coupled device. Hs-cTnI analysis was completed in this format within 25 min, and the results showed a high correlation with those of the CentaurXP® reference system (R(2) > 0.99). The detection limit of the rapid immunosensor was 0.003 ± 0.001 ng/mL cTnI, corresponding to a 10-fold improvement compared to results using the plain enzyme tracer. This demonstrated the measurement of hs-cTnI in a much more cost-effective manner compared to the automated versions currently available.