Detection of animal fat content is necessary to prevent inappropriate product composition labelling measures such as substituting beef with pork or adding lard to other fat-processed products. In this paper, a detection method of animal fat utilizing paper-based biosensors using nano cellulose paper substrate is proposed. The nanocellulose paper substrate was fabricated using bamboo fibre and parenchyma cells. The nanocellulose paper substrate was further decorated with Au nanoparticles to improve the detection’s sensitivity. It demonstrated the measurement of optical properties using UV-vis spectroscopy for several animal fats. It also presented a colorimetric detection using a light source, photodetector, and imaging analysis. It was shown the difference in the color space intensity value before and after the addition of lard samples on AuNP-modified nanocellulose paper. It was reported that the difference in grayscale intensity value of AuNP-modified nanocellulose paper with a reducing agent volume of 3 mL and 4 mL were 37.369 and 55.686, respectively. This demonstrated that the larger the volume of reducing agent added during synthesis, the smaller the size of the nanoparticles and increasing the sensitivity of the sensor. The proposed paper-based biosensor offered a simple fabrication, biodegradable, and can be implemented in a colorimetric imaging system.