In the present paper, the effect of pH value of methyl orange solution on the fluorescence enhancement effect of silver nanoparticles was studied. When pH 1.5 and 2.1 the absorption spectra had little change with added silver colloid. When pH 3.1 a blue shift of 26 nm and a decrease in the intensity of the absorption peak were observed. When the values of pH are in the range of 3.8～8.2 a blue shift of the absorption peak and a 426～456 nm broad absorption band were observed. When silver colloids were added into methyl orange solutions with different pH values the fluorescence intensity of S2→S0 electronic transition decreased and was little impacted by pH value; while the fluorescence intensity of S1→S0 electronic transition was enhanced and was impacted by pH value. The largest and smallest fluorescence enhancement ratios were observed at pH 2.1 and 4.8, respectively. The results indicated that the reasons of effect of pH of methyl orange solution on the fluorescence enhancement of silver colloid depend on methyl orange structure, molecular adsorption mode, media and so on, especially the distance between molecule and silver nanoparticle.