Currently petroleum is being used as a major source for new chemicals, materials & fuels, although it has limitations in availability & costs. It also raises environmental concern. A promising alternative raw material for the production of bioethanol, a fossil fuel substitute, from renewable source is lignocellulosic biomass. Parthenium hysterophorus L, an obnoxious flowering, offers a big challenge to all attempts of control because of its high regeneration capacity, production of huge amounts of seeds, high seed germinabilty & extreme adaptability to wide range of ecosystems. Contact with this plant causes dermatitis & respiratory malfunction in humans, cattle and domestic animals due to the presence of the toxin “parthenin”. In the present work Parthenium is hence chosen for the production of bioethanol. Conversion of hemicellulose, presented in the lignocellulosic biomass, into xylose is one of major steps, as it increases the quantity of reducing sugar which ultimately enhances the ethanol yield through fermentation process followed by saccharification of the available sugar. Hydrolysis and size of the biomass particles plays an important role in xylose production. The objective of this work was to study the effect of particle size on the yield of xylose, which is an important constituent of reducing sugar, from Parthenium hysterophorus during the hydrolysis process under specified conditions. The experiments were carried out with three different particle sizes (0.35 mm, 0.95 mm, 1.78 mm), at different sulphuric acid concentrations and treatment temperatures for 2 h soaking period. Maximum xylose yield was obtained from particle size of 0.95 mm.