open (FILE,"abc345.txt") or die $!;
       while (<FILE>){
       $_ = /((^Query = .*)|(^\>.*)|(Length = .*))/i;
       print "$1\n";
       print "$2\n";
       print "$3\n";
       print "$4\n";
      
   }
  close (FILE) ;
[download]

or

open (FILE,"abc345.txt") or die $!;
       while (<FILE>){
       $_ = /((^Query = .*)|(^\>.*)|(Length = .*))/i;
       print $1, "\n";
       print $2, "\n";
       print $3, "\n";
       print $4, "\n";
      
   }
  close (FILE) ;
[download]

See print for info on proper syntax.

Your code is also missing the match operator. This snippet:

should be changed to:

Otherwise you're trying to assign a value to the $_ variable.

I did whatever you guys said but it is not working, the error is still the same

Suggestions above do work just fine. I made one small addition, if your are on Perl 5.10 you can use the // operator. This sets a default value of "no match" as shown below if say $4 is undefined. There aren't any write statements below so you shouldn't get your reported error anymore. You will however start getting print of undefined value warnings which the "//" avoids.

#!/usr/bin/perl -w
use strict;

open (FILE,"abc345.txt") or die $!;

       while (<FILE>){
       $_ =~ /((^Query = .*)|(^\>.*)|(Length = .*))/i;
       print  $1 // "no match", "\n";
       print  $2 // "no match", "\n";
       print  $3 // "no match", "\n";
       print  $4 // "no match", "\n";   
       }

close (FILE) ;
[download]

For data line of: Query = 345, prints:


Query = 345 
Query = 345 
no match
no match
[download]

Update: Had wrong file handle above in "while (<FILE>)". I used <DATA> to test my code and that didn't get corrected in the original post. Anyway the above code does run on my AS Perl 5.10.X machine. I had thought that perhaps something weird was happening with the "=" as that does have significance in a regex, but only in combination with for example "?=". Result is that you do not have to "escape" the = sign. Above code is fine. An escape "\" is required for these characters:  {}[]()^$.|*+?\

Then I suggest you post your corrected program (with line numbers, so we can see what line the error refers to).
-- 
Ronald Fischer <ynnor@mm.st>

I think You have missed the patten matching operator. 
And with out checking pattern matching status , you are 
seeking for $1,$2,$3,$4 variables. If the matches get 
succeeded only then you can access those variables. 

So you check with if condition 
 
if ( $_ =~ ( regulare expression ) 
{ 
# print the $1,$2,$3,$4 

..... 
}
[download]
 

This is not the problem. The Op's regex guarantees that something of $1,$2,$3,$4 will be undefined even if the true/false sense of a "match" succeeds.

For example, a line can only start with one thing.
e.g, $2 and $3 cannot both be "defined" at the same time...
A line can start with one of these 2 things or something else.
   "Query = "
   ">something"
[download]

The Ops "Length = .*" term will never match unless the line didn't start with Query or > because the .* in those terms will "greedily" gobble up all the characters past those terms in the line. In that case $2 and $3 will be undefined. This is actually ok if that is what was intended.