1. Check the following line is added in your code or not
<%@ Register Assembly="AjaxControlToolkit" Namespace="AjaxControlToolkit" TagPrefix="asp" %>
2. Don't forget to add following line.
<asp:ToolkitScriptManager runat="server"></asp:ToolkitScriptManager>
3. If you are using master page then add script manger to the master.
<asp:ScriptManagerProxy ID="ScriptManagerProxy1" runat="server"></asp:ScriptManagerProxy>