Interstitial fluid (ISF) is a body fluid that fills, surrounds cells and contains various biomarkers, but it has been challenging to extract ISF in a reliable and sufficient amount with high speed. To address the issues, we developed the tilted microneedle ISF collecting system (TMICS) fabricated by 3D printing. In this system, the microneedle (MN) was inserted at 66° to the skin by TMICS so that the MN length could be extended within a safe range of skin penetration. Moreover, TMICS incorporating three MN patches created reliable ISF collecting conditions by penetrating the skin at consistent angle and force, 4.9 N. Due to the MN length increase and the patch number expansion, the surface area of the penetrated tissue was increased, thereby confirming that ISF extraction efficiency was improved. Skin ISF was collected into the paper reservoir on the patch, and the absorbed area was converted into a volume. ISF extraction from the rat skin in vivo by TMICS was well tolerated, and the 2.9 μL of ISF was obtained within 30 s. Therefore, TMICS is promising to apply in the diagnosis of multiple biomarkers in ISF with high speed and stability.