The Gardens at its present site was started in 1859 by a second Agri-Horticultural Society made up of “public spirited citizens”. They acquired a 23-hectare tract of abandoned plantation land at Tanglin from one of their founders, Whampoa (Hoo Ah Kay), and hired Lawrence Niven to create a leisure garden and ornamental park. Lawrence Niven oversaw the landscaping and designed the lay-out of the Gardens in the English Landscape Movement’s style. Many of the features, such as the bandstand hill, ring roads, swan lake and the main gate, remain till today. The Gardens was further expanded by the purchase of two additional tracts of land amounting to 10 hectare in 1866, where the Society built a residential house for its superintendants (later Directors), now called Burkill Hall. In addition to organising flower shows and horticultural fetes, the Society also introduced a zoo to the Gardens, which grew to 140 animals including a leopard, monkeys, deer, kangaroos and emus.

During 1875–78, due to financial difficulties, the Society handed over management and maintenance of the site to the government who then undertook the business of serious reorganisation. The scientific mission of the Gardens evolved when the colonial government assumed management and deployed Kew-trained botanists and horticulturists to administer the Gardens.