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Duke Nukem 3D was originally released for DOS. It was released as a shareware demo (featuring the first three levels of Hollywood Holocaust) and sold on CD.

While it can be emulated, emulation is difficult due to the game's high CPU demands. Windows users should download EDuke32 or another port. These include the shareware data; if you own the game, you can use its data file with the Windows port. To do this, replace the port's DUKE3D.GRP file (usually located in its directory) with the one from an installed DOS copy.

The original DOS version utilized a DOS extender, DOS4GW. As such, no special software is required to run the game.

Duke Nukem 3D requires only a keyboard to operate, but also supports mouse-aim and joystick use. It also supports network usage for online play. The original DOS version used the "Total Entertainment Network", now Pogo, which no longer has game support. Ports include their own network support for IP play.