In this paper, we present a microstructured optofluidic in-fiber Raman sensor for the detection of quinolone antibiotic residue in a water environment based on Ag surface-enhanced Raman scattering (SERS) substrate grown on the surface of the suspended core of micro-hollow optical fiber (MHF). Here, MHF has a special structure with a suspended core and a microchannel inside, which can become a natural in-fiber optofluidic device. Meanwhile, the self-assembled Ag SERS substrate can be grown on the suspended core’s surface through chemical bonds, forming a microstructured optofluidic device with a Raman enhancement effect. Therefore, it can effectively detect the Raman signal of unlabeled trace quinolone antibiotic residue (ciprofloxacin and norfloxacin) inside the optical fiber. The results show that the ciprofloxacin and norfloxacin detection limits (LOD) are 10−10 M and 10−11 M, respectively. Compared with the maximum residue limit (3.01 × 10−7 mol/L) stipulated by the European Union, the results are much lower, and an ideal quantitative relationship can be obtained within the detection range. Significantly, this study provides an in-fiber microstructured optofluidic Raman sensor for the label-free detection of quinolone antibiotic residue, which will have good development prospects in the field of antibiotic water pollution environmental detection. © 2021 Optical Society of America