A single‐step method for isolation of specific cells based on multiple surface markers will have unique advantages because of its scalability, efficacy, and mildness. Herein, we developed multi‐aptamer‐mediated proximity ligation method on live cell membranes that leverages a multi‐receptor co‐recognition design for enhanced specificity, as well as a robust in situ signal amplification design for improved sensitivity of cell isolation. We demonstrated the promising efficacy of our method on differentiating tumor cell subtypes in both cell mixtures and clinical samples. Owing to its simple and fast operation with excellent cell isolation sensitivity and accuracy, this approach will have broad applications in biological science, biomedical engineering, and personalized medicine.

A single‐step method for isolation of specific cells based on multiple surface markers will have unique advantages because of its scalability, efficacy, and mildness. Herein, we developed multi‐aptamer‐mediated proximity ligation method on live cell membranes that leverages a multi‐receptor co‐recognition design for enhanced specificity, as well as a robust in situ signal amplification design for improved sensitivity of cell isolation. We demonstrated the promising efficacy of our method on differentiating tumor cell subtypes in both cell mixtures and clinical samples. Owing to its simple and fast operation with excellent cell isolation sensitivity and accuracy, this approach will have broad applications in biological science, biomedical engineering, and personalized medicine.