The 38-year-old had surgery to remove a tumour last year but was required to continue undergoing treatment for bowel cancer.
Her condition took a turn for the worse in recent weeks and she passed away on Thursday morning.
The long-time couple married last year in the presence of family and friends, including their three-year-old twin sons.
Buffel paid tribute to Stephanie on Instagram. Along with a photo, he wrote: “Our beloved princess has left us this night ... There are no words, she still fought to the end, without ever complaining, she herself was still anxious to get another. We saw her so much. She has inspired us all, and it is now up to us to carry out its positivity further.“
Buffel’s current club Genk were among several sides to offer condolenscences to the midfielder and his family.
A statement read: “With a sad heart KRC Genk announced the death of Stephanie Buysser, wife of Thomas Buffel. Stephanie lost after three years of fighting a long illness and died in the presence of her family at the University Hospital of Leuven.
“The whole family is behind her KRC Genk captain in these difficult days, and expresses its sincere support to him and his family.”
Belgian star Buffel played at Ibrox for three years, under first Alex McLeish and then Walter Smith. He helped the Ibrox club to a league and League Cup double in his first half-season after arriving on a £2.3 million deal from Feyenoord.