Trehalose is a sugar that widely exists in yeast mushroom. It isomerized from Maltose. Trehalose can be used on a food additive. In this work trehalose was studied to evaluate it’s content in 4 type of mushroom. Jew’sear, King oyster, Yanagi mustutake, and Abalone mushroom .The ethanol concentration were varied on 25, 50, 75 and 100 %v/v . The temperature effluents were studied. at room temperature, 50oC and 80oC . Thinlayer chromatography was applied in this work , by compared 2 mobile phase development systems as condition 1 butanol:ethanol:H2O:acetic acid ratio 45:25:15:15 , condition 2 butanol : pyridine : H2O ratio 23.08 : 46.15 : 30.77 The detection system of chromatogram were compared for 2 condition as 20% v/v H2SO4 and 0.5% KMnO4 in 1N NaOH. The trehalose contents were evaluated by UV-visible spectrophotometry compared with standard solution of trehalose. From TLC study the results showed that the mobile phase condition 1 and detection method by spraying with 20% v/v H2SO4 was the best condition. Trehalose exists in all type of mushroom . From the confirmation test, the maximum contents was found in King oyster.