A simple, rapid, and sensitive method using in-tube solid-phase microextraction (in-tube SPME) based on poly(methacrylic acid–ethylene glycol dimethacrylate) (MAA–EGDMA) monolith coupled to HPLC with fluorescence and UV detection was developed for the determination of five fluoroquinolones (FQs). Ofloxacin (OFL), norfloxacin (NOR), ciprofloxacin (CIP), enrofloxacin (ENRO), and sarafloxacin (SARA) can be enriched and determined in the spiked eggs and albumins. CIP/ENRO in eggs and albumins of ENRO-treated hens were also studied using the proposed method. Only homogenization, dilution, and centrifugation were required before the sample was supplied to the in-tube microextraction, and no organic solvents were consumed in the procedures. Under the optimized extraction conditions, good extraction efficiency for the five FQs was obtained with no matrix interference in the process of extraction and the subsequent chromatographic separation. The detection limits (S/N=3) were found to be 0.1–2.6 ng g−1 and 0.2–2.4 ng g−1 in whole egg and egg albumin, respectively. Good linearity could be achieved over the range 2–500 ng mL−1 for the five FQs with regression coefficients above 0.9995 in both whole egg and albumin. The reproducibility of the method was evaluated at three concentration levels, with the resulting relative standard deviations (RSDs) less than 7%. The method was successfully applied to the analysis of ENRO and its primary metabolite CIP in the eggs and albumins of ENRO-treated hens.