Biotin-cellulose nanofiber membranes were developed for use in diagnostic tools. Cellulose acetate (CA) was electrospun into a nonwoven, nanofibrous membrane that was either directly functionalized with propargyl bromide or was first deacetylated (RC) then substituted with propargyl bromide (alkyne-RC). Fourier-transform infrared spectroscopy (FTIR) and Raman spectroscopy were utilized to relate the alkyne peak height ratios to alkyne substitution. The alkyne substitution reaction was dependent on solvent ratio, time, and temperature with optimal reaction conditions of RC in 80/20 (v/v) isopropanol to water at 50°C for 6 hours. Azide-biotin conjugate was “clicked” onto the alkyne-grafted cellulose nanofiber surface via Copper-catalyzed Alkyne-Azide Cycloaddition (CuAAC). FTIR confirmed successful biotin addition. Scanning Electron Microscopy (SEM) verified the nanofiber morphology at each functionalization step. Energy Dispersive X-ray spectroscopy (EDX) mapped the biotin distribution on the membranes. X-ray Photoelectron Spectroscopy (XPS) quantified the total biotin on the nanofiber surface. The biotin-cellulose nanofiber membranes were used in example assays (HABA colorimetric assay and fluorescently-tagged streptavidin assay) where streptavidin specifically bound to the pendant biotin without the need for a blocking agent. The click reaction was specific to only the alkyne-azide coupling and was dependent on pH, ratio of ascorbic acid to copper sulfate (AA:Cu), and time. Copper (II) reduction to copper (I) was successful without use of the ascorbic acid reducing agent, increasing the viability of the click conjugation method with biomolecules. The surface available biotin was found to be dependent on storage medium and time: biotin was unavailable after 24 hours in water but recovered over a period of months with storage in air.