The fluorescent dye Rhodamine-6G (Rh6G) and Rhodamine-B (RhB) doped DNA-CTMA thin films are prepared by spin-coating technology. The quality and optical spectra properties of Rh6G/RhB/DNA-CTMA thin films are characterized by the measurements of surface morphology, UV/vis/infrared absorption spectra and fluorescence spectra. The amplified spontaneous emission (ASE) of dye-doped DNA-CTMA thin films with different dye contents was researched experimentally by pumping of NdYAG pulse laser at wavelength of 532 nm, and is explained theoretically. The experimental results show that the narrowing phenomenon of emission spectra of Rh6G/RhB/DNA-CTMA thin films is very clear and strong. Compared with single dye doped DNA-CTMA thin films, the co-doped Rh6G/RhB/DNA-CTMA thin film is a potential candidate as an organic polymer laser material because it exhibits more wider tuning range of the ASE spectrum.