High‐sensitivity detection of minute quantities or concentration variations of analytes of clinical importance is critical for biosensing to ensure accurate disease diagnostics and reliable health monitoring. A variety of sensitivity‐improving concepts have been proposed from chemical, physical, and biological perspectives. In this review, elements that are responsible for sensitivity enhancement are classified and discussed in accordance with their operating steps in a typical biosensing workflow that runs through sampling, analyte recognition, and signal transduction. With a focus on optical biosensing, exemplary sensitivity‐improving strategies are introduced, which can be developed into “plug‐and‐play” modules for many current and future sensors, and discuss their mechanisms to enhance biosensing performance. Three major strategies are covered: i) amplification of signal transduction by polymerization and nanocatalysts, ii) diffusion‐limit‐breaking systems for enhancing sensor–analyte contact and subsequent analyte recognition by fluid‐mixing and analyte‐concentrating, and iii) combined approaches that utilize renal concentration at the sampling and recognition steps and chemical signal amplification at the signal transduction step.