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Spychecking With Your Brain
How To Help Your Team Spot Spies
Don't behave like a spy.
One of the biggest problems for a defender on the lookout for spies is that team-mates often behave like spies - e.g. running back across the map, or running towards the capture point on an assault map. So don't do it. If you have to do these things, make sure to give your team-mates a clue that you're not a spy, by firing your weapon (in a safe direction), or briefly hitting the voice comms "talk" button. Be careful not to spam with either of these.
Don't look like a spy.
In short, don't run around with your default weapon out unless you have to. This is most applicable to engineers - any good defender is going to get twitchy when he sees an engineer with a railgun running towards a sentry gun. He's not victimising the engineer, he's trying to protect the sentry.
Be a Scout
Despite this being in the TFC manual, many players still seem ignorant: Scouts can remove a spy's disguise by bumping into them (and score a point for doing so). If your team has several sentry guns covering an area, and the enemy is attacking with spies, change class to scout and play tag with any suspicious-looking characters. The spies will soon learn to recognise the sound of sentry guns locking on.
This has a less obvious consequence, too - if you see someone frantically trying to run away from a scout, they may well be a spy.
Spies can unveil other spies, too, but this can be less useful - scouts are fast enough to run down an enemy quickly, while spies will find it much harder to "tag" their victim.