What you've got there is an array reference with a hash reference as its only element. To make an anonymous hash reference, you just use curlies ({}). Since you want to read in multiple comma-delimited rows, you probably want an array of hash references, one hashref for each row.

my @records;    # store the hashrefs in here
my @keys = 
  qw(Emp_No Emp_Lname Emp_Fname Emp_SSN Emp_DOB Emp_aka);

open(IN,"input.txt") or die("Can't open input.txt");
while(<IN>) {
  chomp;

  my %Employee_Rec;
  @Employee_Rec{@keys} = split /,/, $_;   # hash slice

  print "$Employee_Rec{Emp_No}     \n";
  print "$Employee_Rec{Emp_Lname}  \n";
  print "$Employee_Rec{Emp_Fname}  \n";
  print "$Employee_Rec{Emp_SSN}    \n";
  print "$Employee_Rec{Emp_DOB}    \n";
  print "$Employee_Rec{Emp_aka}    \n";

  push @records, \%Employee_Rec;
}
close(IN);
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To inspect @records and make sure you've built it correctly, you can use Data::Dumper.