Protein immobilization on a stationary phase, such as nanocelluloses, is widely used in biodiagnostic, biocatalytic, and bioseparation applications. With the top-down approach which utilizes the native hardwood honeycomb structure, mesoporous cellulose scaffolds can be fabricated without the need for energy-consuming production and bottom-up assembly of nanocelluloses. However, this approach is difficult for preparing softwood-based cellulose scaffolds due to the disintegration of wood cells after complete delignification. Herein, for the first time the use of spruce softwood with a homogenous cellular structure of longitudinally positioned and top-to-bottom joined tracheids is explored as a scaffold for protein immobilization. 1,4-butanediol diglycidyl ether is utilized to crosslink cell wall polysaccharides before the delignification step, thus improving the adhesion between tracheids. The native cellular structure of spruce is well preserved after the complete removal of lignin, enabling the successful production of a highly mesoporous and mechanically robust spruce-derived cellulose scaffold with exceptionally high specific surface area (219 m² g⁻¹). Further amination of the cellulose scaffold allows covalent immobilization of functional biomolecules, such as a lectin protein concanavalin A (Con A) and biotin, on the lumen surfaces and inside the porous cell wall. The Con A immobilized scaffold demonstrates native glycoprotein-binding activity and possible glycoprotein separation application.