This study reports the ageing characteristics of bio-oil and its fractions in presence of three surfactants including Span 80, IGEPAL CO890 and soap that have hydrophilic-lipophilic balance (HLB) values of 4, 17 and 18, respectively. The results show that Span 80 with a low HLB value has not led to phase separation of the bio-oil nor has noticeable effect on ageing characteristics of the bio-oil. IGEPAL CO890 and soap with high HLB values have led to phase separation of the bio-oil into dark and clear fractions. After accelerated ageing, both the bio-oil and the separated bio-oil fractions (dark or clear) have increased viscosities, average molecular weights, water contents and total acid numbers (TANs), e.g. with increases of similar to 34.7%, 16.1%, 1.7% and 6.2% respectively for the bio-oil. Such increases are more in viscosity and average molecular weight (similar to 21.3% and 18.5% vs a maximum of similar to 18.4% and 17.1%) but less in TAN (a maximum of similar to 15.4% vs a maximum of similar to 25.6%) for the dark fractions than the clear fractions. Soap has a more significant effect on bio-oil ageing compared to IGEPAL CO890, due to its basic nature, presence of alkali metals and the separation of fatty acids in bio-oil.