In this work, a two-step method for the extraction of pectin and cellulose fiber from mulberry branch bark, a by-product of sericultural industry, was described. The method was based on the acid extraction of pectin and subsequently alkali treatment for obtaining cellulose fibers. The obtained pectin was high purity with the total galacturonic acid content of 85.46% ± 2.76% and the degree of esterification of 71.13% ± 1.67%. The chemical composition analysis, FTIR spectroscopy, XRD and TG analysis were used to characterize the cellulose fiber at different processing stages. After the two-step chemical process, the cellulose content was increased from 37.38% in original bark to 92.60% in cellulose fiber. The FTIR spectra revealed the removal of pectin, hemicelluloses and lignin from the bark by acid extraction and alkali treatment. The XRD and TG results indicated that the obtained cellulose fibers were with the increased crystallinity and thermal stability, whose crystallinity and degradation temperature were 86.36% ± 5.56% and 355 °C, respectively. This work may provide a new approach for high utilization of mulberry branch bark.