The present invention is directed to the provision of an isotope separation method, which can effectively prevent, without the use of a second gas, a secondary reaction and the formation of a polymer involved in a multiphoton dissociation reaction in laser isotope separation and, at the same time, can efficiently separate a target isotope with low activation energy, and a working substance for use in the isotope separation. The isotope separation method comprises the step of irradiating a working substance for isotope separation comprising a compound represented by formula SiX3-CY2-CZ3 or SiX3-CY═CZ2, wherein X, Y, and Z, which may be the same or different, represent a halogen atom, H, or an alkyl group; and at least one of Z's represents a halogen atom with the remaining Z's being H or an alkyl group, with a laser beam to dissociate only a molecule containing a particular target isotope atom, whereby the dissociation product or the nondissociation molecule is enriched with the target Si isotope atom.