Chimeric chitinase42 (Chit42 containing ChBD) has great potential as a candidate for digesting and recycling chitin as a beneficial nutrient, which can be produced in bioreactors. The plant is one of the most efficient bioreactors that can produce the eukaryotic proteins in active forms. With the plant hairy root system, it is possible to express a variety of recombinant proteins cost-effectively, easily, and quickly. Due to the huge amount of proteins in plants, protein purification can be facilitated by the use of the His-tag. In this research, different computer programs were used for the three-dimensional structural analysis of Chimeric chitinase42 containing His-tag. The results showed that these comparative modeling approaches had a remarkable degree of accuracy in predicting the fused protein structure. The Z-score of -9.38 and -3.64 predicted for Chit42 and ChBD by ProSA represents the good quality of the model. In addition, bioinformatic observations showed that the His-tag was exposed and can be used to purify the Chimeric chitinase42. The Chimeric chitinase42 containing a His-tag was expressed in Nicotiana tabacum hairy roots, and the role of the His-tag in the detection by Western blot and purification using aNi-NTA column was investigated. The presence of the Chimeric chitinase42 was confirmed by analyzing root extracts using SDS-PAGE and Western blot. The purification step was achieved using the His-tag and the Ni-NTA column. The plant-derived Chimeric chitinase42 was confirmed to be biologically active by measuring the chitinase activity of the purified protein on a media plate containing colloidal chitin.