Scoring was difficult at the El Saler course but Uihlein made light of the conditions to shoot a four-under-par 68 for a six-under tally of 138, one ahead of Chile's Felipe Aguilar (71), Frenchman Raphael Jacquelin (66) and Swede Rikard Karlberg (67).
Spanish pair Miguel Angel Jimenez, on his competitive return after breaking his leg in a skiing accident in December, and Alvaro Quiros along with Ryder Cup player Francesco Molinari of Italy were among the casualties of the halfway cut.
Former US amateur champion Uihlein, chasing his first victory as a professional, took control of the tournament towards the end of the day by birdying the 14th, 15th and 17th holes.
"Leading a tournament is fun," the 23-year-old told reporters. "I have been close a couple of times after two rounds so it's nice to have the lead and be able to see how I do.
"It's all part of the learning process and the more I do it the more comfortable I'll feel.
"It's nice to play a course like this now and then where distance isn't at a premium and you have to think your way around," added Uihlein who played a full season on Europe's second tier Challenge Tour last year.
World number 13 Sergio Garcia of Spain was seven strokes adrift of the leader after returning a 73 for 145.
- Sports & Recreation
- Miguel Angel Jimenez
- Felipe Aguilar
- Alvaro Quiros