how to reduce the code

Because you need each test to do something different, there really isn't a lot you can reduce there, except to change the formatting slightly:

if (string.IsNullOrEmpty(TxtUsername.Text))
    {
    LblMessage.Text = "Enter Username";
    }
else if (TxtUsername.Text.Length < 5)
    {
    LblMessage.Text = "Username Should be Minimum 5 character";
    }
else if (!validateStr(mRegExUsername, TxtUsername.Text))
    {
    LblMessage.Text = "Check Note below for guidelines.";
    }
else if (RDBtnSignup.Checked && BLLLoginDetails.Instance.CheckUsernameAvailability(TxtUsername.Text) != 1)
    {
    LblMessage.Text = CISMessages.NotAvailableMsg;
    }
else
    {
    secemail.Visible = !RDIsOwner.Checked;
    e.Cancel = false;
    }

Which is slightly clearer to read.

First, you can remove most of the brackets:

if (string.IsNullOrEmpty(TxtUsername.Text))
    LblMessage.Text = "Enter Username";
else if (TxtUsername.Text.Length < 5)
    LblMessage.Text = "Username Should be Minimum 5 character";
else if (!validateStr(mRegExUsername, TxtUsername.Text))
    LblMessage.Text = "Check Note below for guidelines.";
else if (RDBtnSignup.Checked && BLLLoginDetails.Instance.CheckUsernameAvailability(TxtUsername.Text) != 1)
    LblMessage.Text = CISMessages.NotAvailableMsg;
else
{
    secemail.Visible = !RDIsOwner.Checked;
    e.Cancel = false;
}

But then, it's most likely that this is a body of some event handler and e.Cancel was not assigned in this handler before. If so, it is already null, so you can remove more three lines:

if (string.IsNullOrEmpty(TxtUsername.Text))
    LblMessage.Text = "Enter Username";
else if (TxtUsername.Text.Length < 5)
    LblMessage.Text = "Username Should be Minimum 5 character";
else if (!validateStr(mRegExUsername, TxtUsername.Text))
    LblMessage.Text = "Check Note below for guidelines.";
else if (RDBtnSignup.Checked && BLLLoginDetails.Instance.CheckUsernameAvailability(TxtUsername.Text) != 1)
    LblMessage.Text = CISMessages.NotAvailableMsg;
else
    secemail.Visible = !RDIsOwner.Checked;

But I doubt that it all makes sense, because the code does not look properly designed. We just did not touch your logic, and this is what should really be improved. To improve it more, some information on what do you want to achieve would be needed.

—SA

You can create a function like:

protected string validateData(string _userName)
{
if(_userName == "")
return "Enter Username";
if(another failed condition)
return "Another error message";
.
.
.
.
return "Data Validated";
}

Then check like this:

string _result = validateData(TxtUsername.Text);

if(_result == "Data Validated")
{
proceed with your functionality;
}
else
{
LblMessage.Text = _result;
}

This approach will not only make your code looks clean but also in case of any failed condition, the control will return the error rather than moving forward and check all the remaininng conditions. Just modify the function with your parameters and try.