Cellulose has been one of the most widespread materials due to its renewability, excellent mechanical properties, biodegradability, high absorption ability, biocompatibility and cheapness. Novel, simple and green colorimetric fibrous film sensor was developed by immobilization of urease enzyme (U) and tricyanofuran hydrazone (TCFH) molecular probe onto cellulose nanofibers (CNF). Cellulose acetate nanofibers (CANF) were firstly prepared from cellulose acetate using the simple, green and low cost solution blowing spinning technology. The produced CANF was exposed to deacetylation to introduce CNF, which was then treated with a mixture of TCFH and urease enzyme to introduce CNF-TCFH-U nanofibrous biosensor. CNF were reinforced with tricyanofuran hyrazone molecular probe and urease enzyme was encapsulated into calcium alginate biopolymer to establish a biocomposite film. This CNF-TCFH-U naked-eye sensor can be applied as a disposable urea detector. CNF demonstrated a large surface area and was utilized as a carrier for TCFH, which is the spectroscopic probe and urease is a catalyst. The biochromic CNF-TCFH-U nanofibrous biosensor responds to an aqueous medium of urea via a visible color signal changing from off-white to dark pink. The morphology of the generated CNF and CNF-TCFH-U nanofibrous films were characterized by different analytical tools, including energy-dispersive X-ray patterns (EDX), polarizing optical microscope (POM), Fourier-transform infrared spectroscopy (FT-IR) and scanning electron microscope (SEM). SEM images of CNF-TCFH-U nanofibers demonstrated diameters between 800 nm and 2.5 µm forming a nonwoven fabric with a homogeneous distribution of TCFH/urease-containing calcium alginate nanoparticles on the surface of CNF. The morphology of the cross-linked calcium alginate nanoparticles was also explored using transmission electron microscopy (TEM) to indicate an average diameter of 56-66 nm. The photophysical performance of the prepared CNF-TCFH-U was also studied by CIE Lab coloration parameters. The nanofibrous film biosensor displayed a relatively rapid response time (5-10 min) and a limit of detection as low as 200 ppm and as high as 1400 ppm. Tricyanofuran hydrazone is a pH-responsive disperse dye comprising a hydrazone detection group. Determination of urea occurs through a proton transfer from the hydrazone group to the generated ammonia from the reaction of urea with urease.