put a check on the record already exits on the database.

 protected void Page_Load(object sender, EventArgs e)
        {
            
               if (!string.IsNullOrEmpty(TextBox1.Text))
            {
                SqlConnection con;
                SqlCommand cmd;
                string constr = ConfigurationManager.ConnectionStrings["SomeDataBase"].ToString();
                con = new SqlConnection(constr);

                cmd = new SqlCommand("Select * from Thi where Username=@Username", con);
                con.Open();
                cmd.Parameters.AddWithValue("@Username", TextBox1.Text);
                SqlDataReader dr = cmd.ExecuteReader();
                
                if (dr.HasRows)
                {
                    
                    checkusername.Visible = true;
                    imgstatus.ImageUrl = "Images/NotAvailable.jpg";
                    Label3.Text = "UserName Already Taken";
                    if (Label3.Text == "UserName Already Taken")
                    {
                        TextBox1.Text = " ";
                    }
                    System.Threading.Thread.Sleep(2000);
                }
                else
                {
                    checkusername.Visible = true;
                    imgstatus.ImageUrl = "Images/Icon_Available.gif";
                    Label3.Text = "UserName Available";
                    System.Threading.Thread.Sleep(1000);
                    con.Close();
                }

            }

            else
            {
                checkusername.Visible = false;
            }
}