In the Elizabethan era, either boys, or youth who had not quite entered puberty, played female roles because acting for women was associated with prostitution. Therefore, women didn't act in legitimate theater groups. In Shakespeare's time, Thisbe would have had to be a male. Shakespeare alludes to this in the play when he has Flute worry that his beard will grow in before he can play the part. 

However, Shakespeare also has Flute play the female Thisbe for laughs. For example, when Thisbe (Flute) is coming close to the hole in the wall where he/she meets "her" lover, "she" says "I kiss the wall's hole/not your lips at all," showing that these not-so-polished players are aware that they are performing roles. This reveals that they are not great actors. 

Today, we obviously don't ban women from the stage, but since Flute, the character who plays the female Thisbe, is a male, it makes sense to keep Flute/Thisbe male, so as not to lose the laugh lines.