This way
<asp:TextBox ID="txtEmail" runat="server"></asp:TextBox>
<asp:RequiredFieldValidator ID="RequiredFieldValidator1" ControlToValidate = "txtEmail" Display ="Dynamic" runat="server" ErrorMessage="Please enter email"></asp:RequiredFieldValidator>
<asp:RegularExpressionValidator ID="RegularExpressionValidator1" runat="server" ControlToValidate = "txtEmail" Display ="Dynamic" ValidationExpression="\b[\w\.-]+@((?!gmail|yahoo|).)[\w\.-]+\.\w{2,4}\b"
ErrorMessage="Please enter valid email address. Eg. priyanka@juncturetech.net"></asp:RegularExpressionValidator>
<br />
<asp:Button ID="Button1" runat="server" Text="Submit" />
Ref: http://stackoverflow.com/questions/6548210/block-certain-email-providers-using-regex