Display omitted •Novel, portable, probe-free, liquid cellulose biosensor was explored via a facile one-pot process.•Cellulose biosensor showed specific recognition towards Cu2+ ions within 10 s in water, urine and serum.•Cellulose biosensor displayed excellent sensitivity, selectivity, fast response and biocompatibility. Novel, portable, probe-free, liquid cellulose biosensor was explored via a facile one-pot process for Cu2+ ions detection by naked eyes. The as-prepared biosensor displayed specific recognition towards Cu2+ ions in aqueous solution, even in complex urine and serum samples within 10 s, showing fast response characteristic. There is barely any interference on the Cu2+ ions detection by other metal ions or other complex substances in urine and serum, and the corresponding detection limit is as low as 1.185, 1.9309 and 1.9154 ppm, respectively. Moreover, the cellulose biosensor could act as an excellent candidate to sense Cu2+ ions in biological samples including urine and serum from SD rats with satisfying accuracies and biocompatibility. This study exploits a potential application of cellulose in biosensing and monitoring related to Cu2+ ions.