Stochastic optical reconstruction microscopy (STORM), a super-resolution imaging technology based on immunofluorescence, has gained popularity owing to its straightforward principle, simple optical path, and excellent spatial resolution. However, the enhanced resolution demands greater specificity from antibodies. While direct labeling with primary antibodies is an option, indirect labeling using a"primary + secondary antibody"combination is more commonly employed in practical applications. Considering the issue of species specificity with secondary antibodies, preadsorption is required to increase specificity during production. In this study, we aim to explore the effect of secondary antibody species specificity on dual-color STORM imaging. In the classical erythrocyte skeleton model, based on the mutually exclusive localization between the N-terminal and C-terminal of β-spectrin, we performed two-color STORM imaging of them by labeling them with low- and high-adsorption secondary antibodies, respectively. A comparison with cross-correlation data from simulations revealed that low-adsorption secondary antibodies led to colocalization artifacts in the STORM results. Furthermore, when employing high- and low-adsorption secondary antibodies, two-color STORM results of CD47 and PD-L1 on the MDA-MB-231 cell membrane indicated no colocalization relation between the two proteins. Therefore, this study provides an innovative super-resolution imaging strategy for evaluating secondary antibody species specificity using an erythrocyte skeleton structure model. Additionally, this approach paves the way for accurate identification of biomolecular interactions through dual-color STORM imaging.