French Open champion Stan Wawrinka, the 2014 winner at Melbourne Park, takes on Lukas Rosol as he seeks a 400th tour win which would take him into the last 32 in Melbourne for the fourth time in a row.
In the women`s draw, resurgent two-time champion Victoria Azarenka will look to halt the superb run of 18-year-old Japanese-Haitian Naomi Osaka, who has reached the third round in her Grand Slam debut.
Murray defused Sam Groth, holder of the world`s fastest serve, in the previous round and he is keenly aware of Sousa`s game after playing him twice before at Melbourne Park, winning both times.
However, Portugal`s Sousa also took Murray to four sets at last year`s French Open, and the British second seed knows he will have his work cut out against the world number 33.
“He`s a very good mover. Good athlete. He wins. He knows how to win matches. He understands the game well and he gets the most out of his game,” Murray said.
“He`s the sort of player that if your level`s not quite there, he`ll make it very tough for you, as he did when I played him at the French Open. I was in a bit of trouble against him there.”
Murray has pledged to quit the tournament and rush home if his wife Kim Sears, who is due to give birth to their first child in February, goes into labour.
Switzerland`s Wawrinka has won both his previous matches against fellow 30-year-old Rosol and he will also be confident of reaching the fourth round in his quest for Grand Slam title number three.
Another highlight on the women`s side is former world number one Ana Ivanovic`s clash with Madison Keys, who reached the semi-finals at Melbourne Park last year.