Born in Philadelphia, Pennsylvania, Rubin was educated at Vassar, at Cornell, and at Georgetown University, Washington DC, where she obtained her PhD in 1954. From 1965 she worked at the Carnegie Institution, Washington DC, while also being an adjunct staff member of the Mount Wilson and Las Campanas observatories.

Rubin's main work was primarily concerned with galactic rotation measurements and it led to one of the more persistent problems of modern astronomy. She concentrated on spiral galaxies and measured the rotational velocities of the arms of the galaxy as their distance from the center increases. The velocities of the spiral arms are measured by determining their Doppler shifts. That is light emitted from a body moving away from an observer will show a red shift, and a blue shift when emitted from a body moving toward the observer. The degree of spectral shift is proportional to the velocity of the source.

The initial assumption, based upon Kepler's laws, was that rotational velocity would decrease with distance. Thus the theoretical expectation was that: v ² = GM/r ² where G is the gravitational constant, M the attracting mass, and r the orbital radius. It is clear from the equation that as r increases, v will decrease. Rubin, however, found that the rotational velocity of spiral galaxies either remains constant with increasing distance from the center or rises slightly. The only possible conclusion, assuming the laws of motion, was that the figure for M was too low. But as all visible matter had been taken into account in assessing the mass of the galaxy, the missing mass must be present in the form of ‘dark matter’. Rubin found similar results as she extended her survey. It seemed to her in 1983 that as much as 90% of the universe is not radiating sufficiently strongly on any wavelength detectable on Earth.

Rubin's work presented modern astronomy with two major problems. Firstly to calculate the amount of dark matter in the universe and describe its distribution, and secondly to identify particles that make up the dark matter.

Earlier in her career, in collaboration with Kent Ford, Rubin made the extraordinary discovery that the Milky Way had a peculiar velocity of 500 kilometers per second quite independently of the expansion of the universe. When their results were published in 1975 they were met with considerable skepticism and it was assumed they had miscalculated the distances of the measured galaxies. However, later work by John Huchra and others in 1982 seems to have confirmed their measurements.