Microneedles have recently emerged as a groundbreaking technology in the field of biomedical detection. Notable for their small size and ability to penetrate the superficial layers of the skin, microneedles provide an innovative platform for localized and real-time detection. This review explores the integration of various detection methods with microneedle technology, focusing particularly on its applications in biomedical contexts. First, the common detection methods, such as colorimetric, electrochemical, spectrometric, and fluorescence methods, combined with microneedle technology, are summarized. Then we showcase exemplary uses of microneedle technology in biomedical detection, including the monitoring of blood glucose levels, evaluating infection statuses in skin wounds, facilitating point-of-care testing, and identifying biomarkers in the interstitial fluid of the skin. Microneedle-based detection, with its painless, minimally invasive, and biocompatible approach, holds significant promise for enhancing biological assays. Finally, the review concludes by assessing the future potential and challenges of microneedle detection technology, underscoring its transformative capacity to advance personalized medicine and revolutionize healthcare practices.