Microfluidic chips are in critical demand for emerging applications in material synthesis and biosensing. Herein, we relied on ultrafast laser-processing technology to fabricate a three-dimensional (3D) microfluidic chip, in which semiconducting polymer nanoparticles (SPNs) were continuously synthesized with tunable size and SPN-involved online fluorescence sensing was implemented. A homogeneous distribution of SPNs can be readily realized due to the efficient mixing and powerful vortices of the 3D microfluidic chip, which prevents SPNs from aggregating throughout the synthesis process. Moreover, in the optimized conditions, we unveiled unique SPNs with an ultrasmall particle size (<3 nm) and good monodispersity. By integrating with the high-performance fluorescence of SPNs and 3D microfluidic chip, we further developed an online sensing platform for ratiometric fluorescence assays of H 2 O 2 and oxidase-catalyzed substrates (e.g., glucose), in which a composite of SPNs and neutral red (NR) (SPNs/NR) was used as the mediator. The limit of detection (LOD) for H 2 O 2 is 0.48 μM, and the LOD for glucose is 3.33 μM via the presented platform. This 3D microfluidic synthesis-and-sensing platform provides a new avenue for the facile production of nanoparticles and offers exciting prospects in the field of online sensing biomarkers.