Nanoprobes have been widely used in biomedical engineering. However, antibodies are generally conjugated onto nanoparticles disorderly, which reduces their antigen recognition ability. The existing antibody orientation approaches are usually complex. Here, we developed and demonstrated a simple antibody-oriented strategy for the lateral flow immunoassay of cardiac troponin I by conjugating antibodies onto polystyrene nanospheres at the optimal pH. The binding amount and orientation of antibodies as well as the detection sensitivity were significantly improved. Although pH regulation is commonly used to optimize antibody conjugation, this paper illustrates the mechanism of its antibody orientation enhancement ability for the first time and reveals the important influences of the density, the charge distribution and hydrophilicity of the antibody, the control of the velocities of physical adsorption and chemical coupling, and other factors on antibody orientation. It is of great significance to understand and regulate antibody conjugation on the surface of micro- or nanospheres to construct high-performance probes for in vitro diagnosis applications.