Biochemical analysis by converting biological information into measurable signals is fundamental to life science research, clinical diagnosis, and environmental monitoring. Yet conventional techniques suffer from bulky instrumentation, labor-intensive procedures, and suboptimal sensitivity for low-abundance targets. Microfluidic techniques that precisely manipulate nanoliter-to-picoliter fluids within microscale channels offer a powerful tool for biochemical analysis. By exploiting high surface-to-volume ratio, integration of multiple steps, and enhanced mass and heat transfer, microfluidic devices enable sample-to-answer biochemical assays with improved sensitivity, reduced sample and reagent consumption, and shortened reaction time. This review systematically summarizes recent advances in microfluidic methods for biochemical analysis, with a focus on five major readout modalities including colorimetry, fluorescence, surface-enhanced Raman scattering (SERS), electrochemistry, and mass spectrometry (MS). For each microfluidic method, we overview the underlying working principle and highlight representative applications in biochemical analysis, covering diverse biological targets such as small molecules, proteins, nucleic acids, extracellular vesicles, and cells. The challenges and opportunities in the field of microfluidics for biochemical analysis are also discussed.