Molecular profiling on the single-cell level helps unveil the mystery of gene expression patterns in individual cells at subcellular resolution, enabling discovery of small but meaningful variations that are often overlooked at the population level. Similar to other immunoassays, the most common and economical protocols are developed by combining primary antibodies (1'Abs) and fluorophore-labeled secondary antibodies (2'Abs). The selection of 1' and 2' Abs, however, has been limited by the availability of animal species, consequently resulting in low multiplexing capability. Here we report the development of preassembled Ab pairs using 1'Abs all from the same animal species. We show that multiple molecular targets can be simultaneously labeled without cross reactivity. This simple and general self-assembly technology eliminates the animal species constraints in multicolor immunoassays, offering exciting new opportunities for a wide range of biomedical and clinical applications.