Lateral flow assay (LFA) is an important point-of-care (POC) test platform due to the associated portability, on-site testing, and low cost for diagnosis of pathogen infections and disease biomarkers. However, compared to high-end analyzers in hospitals, LFA devices, in particular, paper-based LFA tests, fall short in accuracy. This study focuses on two ways to improve LFAs: (1) using cellulose fibers, rather than glass fibers for a sample pad, and (2) incorporating a one-step simple, facile, and low cost PVA dam into the LFA. Both strategies (cellulose fiber as a sample pad and water dissolvable PVA dam) contributed to delaying the controlled biomolecule's flow through the nitrocellulose membrane's capillary channels resulting in increased bio-recognition time, thus contributing to the enhancement of LFA sensitivity. PVA modified cellulose fiber-based LFA demonstrated 10 times higher sensitivity than the cellulose fiber-based unmodified LFA, whereas 2 times enhancement was obtained in the cellulose fiber-based sample pad LFA compared to the glass fiber-based sample pad LFA. Ultimately, 20 times increase in sensitivity was achieved in the modified LFA device. This study shows that PVA and eco-friendly cellulose fibers could be incorporated into other paper based POC testing devices for future development.