The novel 3D microfluidic concept of "lab-on-hollow fiber membrane (HFM)" was presented for multifunctional and rapid biological assays, integrating sample size sieving and colorimetric quantification in an HFM. Herein, microporous HFMs with a gradient pore size and high hydrophilic flux were used as microfluidic device substrates. The membrane pores selectively trapped macromolecules within the inner surface, while allowing free diffusion of smaller molecules, including glucose and protein. The microfluidic flow rate in HFM closely followed the Lucas-Washburn and Laplace's models, indicating that the microfluidics facilitated the upward flow of the fluid by microcapillary action without external pumping. Subsequently, for sensing of different biomolecules, a highly sensitive fluorescent or optical chromogenic reagent was immobilized in HFM by an electrostatic interaction. Pyronin G fluorescence reagent was quenched by blood glucose, and the quenching efficiency showed a good linear correlation with glucose concentration (1-22 mM, R2 = 0.997). Moreover, this sensing platform was then further applied for the analysis of urine protein or glucose in the visible spectrum, with a wide testing range. Compared to traditional 2D flat membrane devices, this 3D-HFM microfluidic device exhibited excellent sensing versatility and color rendering uniformity with enhanced sensitivity. Target molecules screening, conditioning, enzymatic recognition, and signal readout of biomolecules have all been implemented on this device, which has paved the way to highly sensitive assays on point-of-care testing (POCT).