In this Letter, we report a combination of non-invasive analysis of the cross-section structure, phase, and chemical composition combining optical coherence tomography (OCT) with spectroscopic methods such as X-ray analytical microscope (μ-XRF) and micro-Raman spectroscopy (μ-RS), which allow us to effectively and conveniently identify the colorants used for each color region and the glass-making process of an ancient multicolored stratified glass eye bead. The results reveal that the sophisticated colors of the glass bead arise from the transition metals and chemical compound crystals deliberately added in the same base glass and carefully adjusted by the glass maker to obtain four colors. We also propose and discuss the provenance of the glass bead. It was probably introduced to China through the Northern Silk Road from Egypt or the Eastern Mediterranean areas about 1400 years ago. The combined multi-analytical technique is the promising approach for precious cultural heritage research.