The in-situ preparation of three-dimensional MOF-199 (3D MOF-199) derived from the electrochemically prepared highly porous nano Cu foam on the surface of a flexible copper wire is reported. The 3D-Cu foam coating was used as a precursor and template for fabrication of MOF-199. The microextraction abil- ity of the in-situ prepared 3D-MOF-199 fiber was evaluated using the prepared fiber for solid phase mi- croextraction (SPME) of selected antibiotics including amoxicillin, azithromycin, ciprofloxacin, cefixime and gentamicin coupled to high-performance liquid chromatography with UV detection. Under the opti- mized condition, the calibration curves were linear in the range of 1–100 ìg L .1 (r 2 above 0.9921) for both water and urine matrices. Limits of detection and limits of quantification were 0.14–0.62 ìg L .1 and 0.53–2.17 ìg L .1 in the selected matrices, respectively. In addition, the repeatability of the method was evaluated by considering the relative standard deviation (RSD%). The intra-day and inter-day RSDs of the method with the single fiber was in the range of 2.8% to 4.9% and from 3.1% to 4.9%, respectively. Fur- thermore, the fiber-to-fiber reproducibility ranged from 2.9% to 5.5%. The enrichment factors were also in the range of 32 to 55. Finally, the method was successfully used for analysis of amoxicillin, azithromycin, ciprofloxacin, cefixime and gentamicin in urine samples. Relative recoveries for spiked urine samples were in the range of 90–105%.