Base-pairing interactions between nucleic acids mediate target recognition in many biological processes. We developed a super-resolution imaging and modeling platform that enabled the in vivo determination of base pairing-mediated target recognition kinetics. We examined a stress-induced bacterial small RNA, SgrS, which induces the degradation of target messenger RNAs (mRNAs). SgrS binds to a primary target mRNA in a reversible and dynamic fashion, and formation of SgrS-mRNA complexes is rate-limiting, dictating the overall regulation efficiency in vivo. Examination of a secondary target indicated that differences in the target search kinetics contribute to setting the regulation priority among different target mRNAs. This super-resolution imaging and analysis approach provides a conceptual framework that can be generalized to other small RNA systems and other target search processes.