In the fight against the worldwide pandemic coronavirus disease 2019 (COVID-19), simple, rapid, and sensitive tools for nucleic acid detection are in urgent need. PCR has been a classic method for nucleic acid detection with high sensitivity and specificity. However, this method still has essential limitations due to the dependence on thermal cycling, which requires costly equipment, professional technicians, and long turnover times. Currently, clustered regularly interspaced short palindromic repeats (CRISPR)-based biosensors have been developed as powerful tools for nucleic acid detection. Moreover, the CRISPR method can be performed at physiological temperature, meaning that it is easy to assemble into point-of-care devices. Microfluidic chips hold promises to integrate sample processing and analysis on a chip, reducing the consumption of sample and reagent and increasing the detection throughput. This review provides an overview of recent advances in the development of CRISPR-based biosensing techniques and their perfect combination with microfluidic platforms. New opportunities and challenges for the improvement of specificity and efficiency signal amplification are outlined. Furthermore, their various applications in healthcare, animal husbandry, agriculture, and forestry are discussed.