Khenissi, who was top scorer in last year's African Champions League, injured his thigh playing for his club Esperance last week and has been ruled out for a month.
Tunisia have already lost talisman Youssef Msakni, who underwent knee surgery in the U.S. last week and faces a lengthy period on the sidelines.
However, Mohamed Amine Ben Amor, who was also a doubt after suffering a knee injury in an international friendly against Iran in March, makes the preliminary squad as the north Africans hope he can be fit in time for the tournament in Russia.
Tunisia open their World Cup campaign against England in Group G before taking on Belgium and Panama. All 32 nations at the tournament must submit a final 23-player squad to FIFA by June 4.
Squad: Goalkeepers: Moez Ben Cherifia (Esperance), Farouk Ben Mustapha (Al Shabab), Moez Hassen (Chateauroux), Aymen Mathlouthi (Al Baten).
Defenders: Rami Bedoui (Etoile Sahel), Yohan Benalouane (Leicester City,), Syam Ben Youssef (Kasimpasa), Dylan Bronn (Ghent), Khalil Chammam (Esperance), Oussama Haddadi (Dijon), Ali Maaloul (Al Ahly), Yassine Meriah (CS Sfaxien), Bilel Mohsni (Dundee United), Hamdi Nagguez (Zamalek).
Midfielders: Anice Badri (Esperance), Mohamed Amine Ben Amor (Al Ahli Riyadh), Ghaylene Chaalali (Esperance), Ahmed Khalil (Club Africain), Saifeddine Khaoui (Troyes), Mohamed Wael Larbi (Tours), Karim Laribi (Cesena, Italie), Ferjani Sassi (Al Nasr), Ellyes Skhiri (Montpellier), Naim Sliti (Dijon), Bassem Srarfi (Nice).
Forwards: Ahmed Akaichi (Al-Ittihad), Fakhreddine Ben Youssef (Al Ittifaq), Saber Khalifa (Club Africain), Wahbi Khazri (Stade Rennes).
(Reporting by Mark Gleeson in Johannesburg; Editing by John O'Brien)
