The spontaneous haemolytic activity has been used as an indicator of the effects of inherent or external factors on the immune system and the disease resistance of fish. The present study was undertaken to estimate the spontaneous haemolytic activity of healthy rohu, (Labeo rohita) fingerlings serum and to determine optimum temperature, capacity to lyse RBC from different donors, heat sensitivity, storage temperature and effect of different agents like EDTA, EGTA, on spontaneous haemolytic activity of rohu. Besides this, complement binding factors such as zymosan, Lipopolysacharide were tested for spontaneous haemolytic 50 % (SH 50%) activity. The effect of bacterial culture was also determined on SH 50% activity of rohu sera. The optimum lysis was obtained with rabbit RBC at 20 °C for rohu sera. The heat sensitivity of sera found to be 65 °C. The haemolytic activity of rohu sera maintained fairly good activity after short term storage at -80 °C as well as 5 months storage at -80 °C. The EGTA inhibited the SH 50% but EDTA enhanced the SH 50% activity. Zymosan significantly inhibited the haemolytic activity of rohu sera whereas bacterial LPS and Aeromonas hydrophila culture reduced the SH 50% activity to less extent.