The conjugated polymer poly {[2-[2′,5′-bis(2″-ethylhexyloxy)phenyl]-1,4-phenylenevinylene]-co-[2-methoxy-5-(2′-ethylhexyloxy)-1,4-phenylenevinylene]} (B-co-MP) has been proven to be an excellent laser medium with a high photochemical stability. Moreover, the impact of γ-irradiation on its optical and chemical properties has been investigated. Herein, the spectral and amplified spontaneous emission (ASE) characteristics of B-co-MP at various concentrations under γ-irradiation doses are studied. Various concentrations of B-co-MP in tetrahydrofuran (THF) were prepared. The samples were irradiated with various γ-doses from 5 to 20 kGy using a Co-60 source at room temperature. The absorption, fluorescence, and ASE spectra were dramatically blue-shifted after the γ-irradiation. This indicates that the increment in the γ-irradiation dose led to a widening in the energy gap and reduction in the number of carbon atoms (N). The change in the spectral profiles could be attributed to chain conformational alterations and/or chain scission induced by the γ-irradiation. We anticipate this study to boost our understanding of optical and structural profiles of B-co-MP under various conditions, including γ-irradiation and the potential utility of this copolymer in a variety of applications.