In recent years, infectious diseases caused by drug-resistant bacteria have been expanding worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) is a type of drug-resistant bacterium that causes nosocomial infections. To detect nosocomial infections at an early stage, it is important to develop a rapid and simple method to detect MRSA. In this study, we developed a simple colorimetric biosensor that separates target DNA-probe-modified gold nanoparticles (AuNPs) using a freeze-dried Ca2+-cross-linked 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO)-oxidized cellulose nanofiber (TOCN) filter. First, the AuNP probe was hybridized with the target ssDNA to form a complex. Because of the large size of the DNA–AuNP probe complex, it was trapped by the filter and showed the red color characteristic of AuNPs. The color intensity of the AuNPs increased with the DNA concentration. The color filter sensor was able to quantify the target DNA in the range of 10–1000 pM without polymerase chain reaction (PCR) amplification (3.0 × 108–3.0 × 1010 copies). The sensor we developed can filter out unreacted AuNP probes, thus eliminating the need for bond/free (B/F) separation. Our sensing system was able to finish detection in 1 min and was selective enough to distinguish between MRSA and S. aureus (SA).