Abstract The traditional way of disease diagnosis is to detect biomarkers in serum, blood, urine, and saliva, but it often faces wound infection and pain, and usually requires professional medical personnel to operate, which is unacceptable for most people. As a result, minimally invasive detection of biomarkers has attracted wide attention and is expected to become a new method for patient monitoring and disease diagnosis. Interstitial fluid (ISF) acts as a medium between cells and the circulatory system, serving as a reservoir of biomolecules, nutrients, and metabolic products. Microneedles (MNs) are effective carriers for delivering drugs, biomolecules, vaccines, and stem cells. What's more, MNs provide a painless, simple, and minimally invasive method to collect skin ISF. In summary, the structural and functional characteristics of MNs provide incomparable advantages and open the door for future diagnosis and monitoring. MNs techniques are minimally invasive, convenient, and patient compliant for individualized and long‐term treatments, which open up new areas of disease diagnosis. In this review, various MNs techniques for ISF sampling in disease diagnosis are reviewed, and their future challenges and development prospects are discussed.