The 21-year-old, who joins for an undisclosed fee, has penned a three-year deal at the Amex Stadium and becomes Brighton's seventh signing of the summer.
Seagulls boss Chris Hughton told the official club website: "Ales already has good experience playing regular senior football in his homeland - and has played Europa League football as well as qualification phases in the Champions League.
"He has good experience for his age and the stage of his career he is at. We are delighted to welcome him to the club and the plan now is to break into the first-team group.
"Initially we will monitor his progress in training over the coming weeks, and then make a decision in terms of what is best for Ales's development - and one of the options might be for him to go on loan in order to continue playing regular senior football."
Mateju, who began his professional career at hometown club FK Pribram, joined Plzen in 2015 and helped them win the Czech First League title during his first season at the club.