Hemp fibers, recognized for their breathability, specific strength, and ultraviolet resistance, are widely utilized in textile manufacturing and composite materials. Bio-degumming is a promising alternative technology to traditional chemical degumming that can be used to produce hemp fibers due to its eco-friendly nature. However, its lower efficiency has hindered its widespread adoption. The unclear and complex structure of the gums leads to a poor understanding on the enzyme types required for bio-degumming, thereby restricting improvements in its efficiency. In this study, the morphological characteristics, polysaccharide composition, and branched structure of hemp stem, roving fibers, and refined fibers were investigated using scanning electron microscopy and laser scanning confocal microscopy in combination with immunofluorescence techniques, with a view to identify the enzymes necessary for the efficient bio-degumming of hemp. The results revealed that the gums were primarily located in the middle lamella, phloem parenchyma, and certain xylem tissues. These tissues showed chunk-like, fence-like, and plate-like shapes, respectively, and tightly wrapped around the fiber bundles. In these tissues, pectin comprised low-esterified homogalacturonan, along with rhamnogalacturonan carrying galactan and arabinan branches. Xylan exhibited acetyl, arabinose, and glucuronic acid branches, while mannan displayed acetyl and galactose branches. Partial xylan and mannan were masked by pectin, and the branching structures impeded their enzymatic removal. As a consequence, the necessary enzymes and their synergistic effects for effective hemp roving degumming were elucidated. Pectin degradation was facilitated by pectate lyase and rhamnogalacturonan-degrading enzymes. Xylan and mannan were effectively removed by endo-xylanase and endo-mannanase, a process necessitating the synergistic action of branched-chain-degrading enzymes, including the esterase, α-L-arabinofuranosidase, α-galactosidase, and α-glucuronidase. This study provided practical strategies to enhance the efficiency of hemp bio-degumming.