In this study, we present a disposable and inexpensive paper-like gold nanoparticle-embedded cellulose nanofibril substrate for the rapid enumeration ofEscherichia coli(E. coli) using surface-enhanced Raman scattering (SERS) mapping. A disposable SERS substrate was simply constructed by mixing CNF and gold chloride solution at 120 degrees C in a water bath. The application of the resulting substrate was carried out by enrichment and SERS detection ofE. coli. To this end, the spherical gold nanoparticle-embedded cellulose nanofibril substrate was used as a scavenger forE. coli. After the target bacteriaE. coliwere separated from the matrixviaoriented antibodies, the sandwich assay procedure was carried out using 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB)-coated Au nanorod particles that acted as SERS mapping probes. The distribution density of DTNB was demonstrated visually using SERS mapping, and the assay was completed in one hour. The correlation between theE. coliand SERS mapping signals was found to be linear within the range of 15 cfu mL(-1)to 1.5 x 10(5)cfu mL(-1). The limit of detection for the SERS mapping assay was determined to be 2 cfu mL(-1). The selectivity of the developed method was examined withMicrococcus luteus(M. luteus),Bacillus subtilis(B. subtilis), andEnterobacter aerogenes(E. aerogenes), which did not produce any significant response. Furthermore, the developed method was evaluated for detectingE. coliin artificially contaminated samples, and the results were compared with those of the plate-counting method.