Reus has scored 17 goals and created 11 more for the Bundesliga leaders this season, but is absent for the last 16, first leg against Spurs at Wembley with a thigh strain.
"Now he's also going to be missing for the important game against Tottenham - that won't make it any easier," said former Germany captain Matthaeus, now a Sky pundit.
"The third-best team in the Premier League against the best in the Bundesliga - I see the chances as 50/50.
"In Marco Reus, Dortmund are missing their best player."
However, it is not just Reus who will be missing against Spurs.
Right-back Lukasz Piszczek is out with a heel injury, stand-in centre-back Julian Weigl has flu and striker Paco Alcacer is sidelined by a shoulder injury.
Reus injured his thigh last Tuesday while drilling home a direct free-kick against Werder Bremen, but Dortmund lost on penalties as the German Cup tie finished 3-3 after extra time.
Spurs are also without big names of their own as England forwards Harry Kane and Delle Alli are injured.
Matthaeus says Dortmund's 3-3 draw at home to Hoffenheim on Saturday showed how much they miss Reus' influence and organisation.
In his absence, Jadon Sancho, 18, already a full England international, has taken over playmaking duties and was outstanding in scoring a goal, setting up two more and hitting the post against Hoffenheim before Dortmund leaked three late goals.
"He (Reus) is the one who instills respect until the very end and has a controlling hand on the whole Dortmund game," added Matthaeus.
"They have also won games without him and were 3-0 ahead on Saturday."
Matthaeus says Saturday's draw will have given hope to defending champions Bayern Munich, who closed the gap in the Bundesliga table to five points behind Dortmund with a 3-1 home win against Schalke on Saturday.
"The points Dortmund lost will have given Bayern a push, they'd have been inspired."