Nature abounds with mysterious biological creatures and organisms that exhibit unique surface wettability, such as lotus leaves with the self-cleaning property, rice leaves and butterfly wings with directional adhesion property, the mosquito eyes with antifogging functionality, and the Namib desert beetle and spider silk with water collection ability. Here, a full-range droplet manipulation system with a extreme wetting substrate is developed for the multiplex bioassay of a single-droplet sample. To fabricate the extreme wetting substrate, adhesive and superomniphobic (SPO) nanoparticles are sequentially sprayed onto a stretchable polymer substrate and then subjected to oxygen plasma treatment to form superhydrophilic (SPI) patterned regions. The SPO-layer-coated substrate maintains its extreme liquid repellency even under high strain (100%) and repeated cyclic stretching and releasing (1000 cycles). Thus, various liquid droplets can be precisely manipulated and dispensed onto the pre-defined SPI patterns on the SPO PDMS by using the developed droplet manipulation system. This system enables a multiplex colorimetric bioassay capable of detecting multiple analytes, including glucose, uric acid, and lactate, from a single droplet of the sample. In addition, the detection of glucose concentration in a plasma droplet of diabetic mouse demonstrates the feasibility of the proposed system for efficient clinical diagnostic applications.