Matrix certified reference materials (CRMs) are an indispensable part of method validation and have played an important role in ensuring reliable analytical results. To retain similarity to real samples, a new matrix CRM for the mass fraction of ciprofloxacin in whole liquid egg was developed by use of incurred materials with a target value corresponding to residue levels in real sample. The source materials were collected from laying hens following oral administration of ciprofloxacin. An optimized homogenization method and a strict bottling process were applied to bulk whole egg materials to prepare the CRM candidate. The mass fraction of ciprofloxacin in whole liquid egg was certified by a collaborative characterization program with eight accredited participating laboratories. Liquid chromatography coupled with isotope dilution mass spectrometry was studied as a reliable reference method for value assignment and was used by all participating laboratories. The certified value and expanded uncertainty (k = 2, at a confidence level of 95%) was 39.7 ± 5.2 μg/kg for ciprofloxacin in whole liquid egg. Homogeneity, long-term stability at -70 °C for 12 months, and short-term stability at -18 °C, 4 °C, and room temperature were assessed for 9 days. Additionally, uncertainties arising from inhomogeneity, instability, and characterization were analyzed in detail and fully estimated. This CRM would be a useful tool for validation of analytical methods and proficiency testing in ciprofloxacin residue analysis of egg. Graphical Abstract.