Fluorescence behavior of poly(3-alkylthiophenes) (P3ATs) in poly(methyl methacrylate) (PMMA) films was studied under tensile loadings. The elastic deformation of the matrix induced by small strains up to 0.2% had a greater influence on time-resolved fluorescence rather than the steady-state one from P3ATs. The decay time of fluorescence decreased linearly as the applied stress was increased, and reached a minimum. We maintained that the attractive phenomenon for the time-resolved fluorescence under the tensile loadings was associated with distortion or deformation of the π-conjugated structure in P3AT chains having large molecular weight. When the applied strain exceeded the elastic limit of the matrix, the time-resolved or steady-state fluorescence behavior was unchanged. However, large elongation over 10% led to the red-shift of the steady-state fluorescence maximum and an increase in the decay time. These fluorescence properties, indicating the growth of the π-conjugation, were mainly governed by strain.