Accurate, sensitive, and highly selective detection of biological targets is of great significance in a wide range of domains, including clinical diagnosis, disease therapy, drug development, environmental monitoring, and food safety control. Recently, with advances in microfabrication and nanotechnology, the combination of nanocatalysis and microfluidics has achieved great success in efficient bioanalysis benefiting from both components. This review aims to summarize the recent developments in nanocatalysis-assisted bioanalytical microfluidic devices involving extensively studied catalytically active nanomaterials, widely used microfluidic approaches, commonly adopted detection techniques, and their analytical applications for various biological targets, including small molecules, proteins, nucleic acids, cells, and bacteria. In addition, the benefits from integrated nanocatalysts, including signal conversion and signal amplification toward the improvement of detection sensitivity, are also highlighted. Ultimately, we discuss the key challenges and future development trends of nanocatalysis-assisted microfluidics, expecting to provide guidance for point-of-care testing devices suitable for practical use.