Aluminum and copper are fairly abundant heavy metals used in several industries, such as pharmaceuticals, food packaging, and kitchen utensil manufacturing industries, and cause emerging health issues due to their toxic, bioaccumulative, and nonbiodegradable nature. Thus, heavy metal detection is a vital solution to mitigate these hazardous health issues. This study demonstrates the development of folic acid carbon quantum dot (CQD)-loaded chitosan-based thin-film biosensors comprising electrostatically adsorbed glutamate dehydrogenase (GDH) and glucose oxidase (Gox) enzymes. They were characterized using various techniques such as TEM, XRD, SEM, and XPS and tested for aluminum and copper detection using a fiber-optic spectrometer device coupled to a reflectance probe. The biosensing results showed that aluminum and copper ions can be detected with a response time of 2 min and limits of detection of 24 and 19 ppb, respectively, in a concentration range of 0−100 μM. The accuracy of detection was found to be in the range of 100−102%. A microplasma atomic emission spectroscopy (MP-AES)-based method was also used as a standard sophisticated equipment reference for estimation. Results clearly indicate that GDH/Gox-CQD-loaded chitosan-based thin-film biosensors coupled to the fiber-optic device show tremendous potential to function as a tool for real-time monitoring of aluminum and copper in various water resources.