Khoo Cheng Lim was born in 1808 in Fujian, China to Khoo Wat Seng. Khoo Wat Seng was among the early Chinese settlers in Penang and was the co-founders of the Khoo family clan temple, Ee Kok Tong in 1835 (later known as Leong San Tong Khoo Kongsi).
Khoo Cheng Lim who was Khoo Wat Seng's eldest son, was first married Lim Neo in China, in which he had two sons, Khoo Soo Chuan and Khoo Soo Teong. He later moved to Penang to join his father. In Penang, he married Koh Keng Yean (辜輕煙) daughter of Koh Kee Jin. The Koh family was a well established member in Penang and its patriarch Koh Lay Huan was the Kapitan of Penang. The marriage was arranged so as to increase the power of the Koh-Khoo families in the Straits Settlements.
Khoo Cheng Lim had four sons through Koh Keng Yean, and his youngest son, Khoo Cheow Teong was a Chinese Kapitan of Asahan, and was made a Justice of Peace by the British in Penang. Khoo Cheng Lim's youngest son through his principal wife in China, Khoo Soo Teong was born in 1883, he married Quah Neo in China and had four sons. His second son, Khoo Ban Seng later moved to Penang and worked for his uncle, Khoo Cheow Teong. Khoo Ban Seng married Yeoh Cheam Neo (d. 1939) and had a son, Khoo Ewe Aik.